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Purinergic Regulation of Breathing in Cane toads (Bufo marinus)
by
Joseph Chau
A thesis submitted in conformity with the requirements for the degree of Master of Science
Cell and Systems Biology
University of Toronto
© Copyright by Joseph Chau (2014)
ii
Purinergic Regulation of Breathing in Cane toads (Bufo marinus)
Joseph Chau
Master of Science
Graduate department of Cell and Systems Biology
University of Toronto
(2014)
Abstract
Previous studies have shown that descending inputs from the midbrain exert an inhibitory
modulation on fictive breathing frequency in the cane toad (Bufo marinus) and that these inputs
become strengthened during chronic hypoxia (CH). I hypothesize that adenosine (ADO) plays a
role behind this inhibitory modulation, due to the fact that (1) ADO is a naturally circulating
metabolite, (2) extracellular [ADO] increases significantly during CH, and (3) the adenosine 1
receptor (A1R) is the primary receptor for ADO in the brain. In this study breathing was
measured by recording motor output (fictive breathing) from respiratory nerves in an isolated
brainstem-spinal cord preparation superfused with artificial cerebral spinal fluid (aCSF). The
results indicate that ADO inhibited fictive breathing frequency (fR) and total fictive ventilation
(TFV) and that this inhibition was mediated primarily by the A1R. Transecting the midbrain
caused an increase in fictive fR and TFV at hypercapnic aCSF pH levels and reduced the
inhibitory modulation mediated by A1R such that A1R activation by endogenous ADO no longer
influenced breathing. Collectively, the study supports a role for ADO as a factor behind the
inhibitory modulation on breathing exerted by descending input from the midbrain.
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Table of Contents
List of Abbreviations .......................................................................................................................... vii
List of Figures ....................................................................................................................................... x
Chapter 1: General Introduction .......................................................................................................... 1
1.1 Breathing in Animals ............................................................................................................... 2
1.2 Breathing in Amphibians ......................................................................................................... 2
1.3 Breathing Patterns in Amphibians .......................................................................................... 4
1.4 Control of Breathing ................................................................................................................ 5
1.4.1 Central Processes ......................................................................................................... 5
1.4.2 Olfactory Chemoreceptors .......................................................................................... 6
1.4.3 Pulmonary Stretch Receptors ..................................................................................... 6
1.4.4 Peripheral O2/CO2/pH sensitive chemoreceptors ...................................................... 7
1.4.5 Central pH/CO2 sensitive chemoreceptors................................................................. 7
1.4.6 Neurotransmitters ........................................................................................................ 8
1.5 In vitro Brainstem – Spinal Cord Preparation ........................................................................ 8
1.6 Adenosine ................................................................................................................................. 9
1.7 Hypothesis and Objectives .................................................................................................... 10
Chapter 2: General Materials and Methods ...................................................................................... 12
2.1 Experimental Animals ........................................................................................................... 13
2.2 Artificial Cerebral Spinal Fluid (aCSF) Solution ................................................................ 13
2.3 The In vitro Brainstem-Spinal Cord Preparation ................................................................. 14
2.4 Experimental protocol: aCSF pH Changes and Treatment with Adenosine, CCPA or
DPCPX ................................................................................................................................... 15
2.5 Experimental protocol: Midbrain Transection ..................................................................... 17
2.6 Controls .................................................................................................................................. 18
iv
2.7 Data and statistical analyses .................................................................................................. 18
Chapter 3: The Effects of Adenosine, CCPA and DPCPX on intact Brainstem-Spinal Cord
Preparations ................................................................................................................................... 20
3.1 Introduction ............................................................................................................................ 21
3.2 Hypothesis & Objectives ....................................................................................................... 22
3.3 Materials and Methods .......................................................................................................... 24
3.4 Data Analysis ......................................................................................................................... 26
3.5 Results..................................................................................................................................... 27
3.5.1 Fictive Breathing Frequency ..................................................................................... 27
3.5.2 Fictive Episodes per Minute ..................................................................................... 29
3.5.3 Fictive Breaths per Episode ...................................................................................... 31
3.5.4 Integrated Area of Fictive Breaths ........................................................................... 33
3.5.5 Fictive Breath Duration ............................................................................................. 35
3.5.6 Total Fictive Ventilation ........................................................................................... 37
3.6 Discussion............................................................................................................................... 39
Chapter 4: The effects of a Midbrain Transection on Fictive Breathing ........................................ 41
4.1 Introduction ............................................................................................................................ 42
4.2 Hypothesis & Objectives ....................................................................................................... 43
4.3 Materials and Methods .......................................................................................................... 45
4.4 Data Analysis ......................................................................................................................... 46
4.5 Results..................................................................................................................................... 47
4.5.1 Fictive Breathing Frequency ..................................................................................... 47
4.5.2 Fictive Episodes per Minute ..................................................................................... 49
4.5.3 Fictive Breaths per Episode ...................................................................................... 51
4.5.4 Integrated area of fictive breaths .............................................................................. 51
4.5.5 Fictive Breath Duration ............................................................................................. 51
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4.5.6 Total Fictive Ventilation ........................................................................................... 55
4.6 Discussion............................................................................................................................... 57
Chapter 5: Effects of Adenosine, CCPA and DPCPX on Fictive Breathing Following a
Midbrain Transection .................................................................................................................... 60
5.1 Introduction ............................................................................................................................ 61
5.2 Materials and Methods .......................................................................................................... 63
5.3 Data Presentation and Analysis ............................................................................................. 64
5.4 Results..................................................................................................................................... 66
5.4.1 Fictive Breathing Frequency ..................................................................................... 66
5.4.2 Fictive Episodes per Minute ..................................................................................... 70
5.4.3 Fictive Breaths per Episode ...................................................................................... 73
5.4.4 Integrated Area of Fictive Breaths ........................................................................... 76
5.4.5 Fictive Breath Duration ............................................................................................. 79
5.4.6 Total Fictive Ventilation ........................................................................................... 83
5.5 Discussion............................................................................................................................... 86
5.5.1 Possible Sites of Adenosine Action.......................................................................... 86
5.5.2 The Effects on Adenosine, CCPA and DPCPX ...................................................... 87
Chapter 6: Summary, Conclusions and General Discussion ........................................................... 91
6.1 Summary of the Major Results of the Thesis ....................................................................... 92
6.2 The Major Conclusions of the Thesis ................................................................................... 93
6.3 The Experimental Approach and Manipulation of Adenosine Receptors .......................... 94
6.4 The Effects of pH and Adenosine on Respiratory-Related Motor Output ......................... 95
6.5 Chronic Hypoxia and Adenosine .......................................................................................... 96
6.6 Experimental Limitations & Future Suggestions ................................................................. 97
6.7 Conclusion ............................................................................................................................ 100
References ......................................................................................................................................... 101
vi
Appendix ........................................................................................................................................... 112
A1. Summary of the Experimental Protocol and an Explanation of the Data Contained
Within the Appendix ............................................................................................................ 113
A2. Data Analysis ....................................................................................................................... 116
A3. Results................................................................................................................................... 117
A3.1 Fictive Breathing Frequency ................................................................................... 117
A3.2 Fictive Episodes per Minute ................................................................................... 118
A3.3 Fictive Breaths per Episode .................................................................................... 119
A3.4 Total Fictive Ventilation Index............................................................................... 120
A3.5 Integrated Area of the Fictive Breaths ................................................................... 121
A3.6 Fictive Breath Duration ........................................................................................... 122
A3.7 Time.......................................................................................................................... 123
A3.8 Midbrain Transection .............................................................................................. 124
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List of Abbreviations
A1R: adenosine A1 receptor
A2AR: adenosine A2A receptor
A2BR: adenosine A2B receptor
A3R: adenosine A3 receptor
AC: adenylate cyclase
aCSF: artificial cerebral spinal fluid
ADO: adenosine
AMP: adenosine monophosphate
AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
ATP: adenosine tri-phosphate
Ca2+
: calcium
CaCl2: calcium clhloride
cAMP: cyclic adenosine mono-phosphate
CCPA: 2-chloro-N(6)-cyclopentyladenosine
CH: chronic hypoxia
CL-IB-MECA: 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
CNS: central nervous system
CO2: carbon dioxide
CSF: cerebral spinal fluid
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CRG: central rhythm generator
DPCPX: 8-Cyclopentyl-1,3-dipropylxanthine
ECD: excitotoxic cell death
eng X: raw electroneurogram
fR: fictive breathing frequency
GABA: gamma-aminobutyric acid
Gi: inhibitory G proteins
GIRK: G protein-gated inwardly rectifying K+ channels
Gs: stimulatory G proteins
H+: hydrogen
I-neuron: inspiratory neuron
K+: potassium
KCl: potassium chloride
LC: locus coeruleus
MCT: multiple comparison test
MgCl2: magnesium chloride
MS222: 3-aminobenzoic acid ethyl ester
NaCl: sodium chloride
NaHCO3: sodium bicarbonate
NI: nucleus isthmus
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NMDA: N-methyl-D-aspartate
NO: nitric oxide
O2: oxygen
PCO2: partial pressure of carbon dioxide
pFRG: parafacial respiratory group
PKC: protein kinase C
PO2: partial pressure of oxygen
PreBötzC: Pre-Bötzinger complex
PSR: pulmonary stretch receptors
RT-PCR: reverse transcription polymerase chain reaction
S.E.M: Standard Error of the Mean
TFV: total fictive ventilation
Vth: trigeminal nerve root
VIIth: facial cranial nerve
IXth: glossopharyngeal cranial nerve
Xth: vagus nerve
∫eng X: integrated electroneurogram
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List of Figures
Figure 1.1: The respiratory cycle of the adult anuran amphibian.
Figure 2.1: Schematic diagram of the apparatus used to measure brain activity from isolated
cane toad brainstem preparations.
Figure 2.2: Flow chart illustrating the general steps taken within the following study.
Figure 2.3: A lateral view of the toad brain illustrating the transected areas (dotted lines) used in
the current study.
Figure 2.4: Pyramid illustrating the relationship between respiratory variables evaluated in the
current study.
Figure 3.1: Overview of the stages and general procedures taken within the experiment on intact
brainstem preparations.
Figure 3.2: The effects of ADO and A1R analogs on fictive breathing frequency in intact
brainstem preparations.
Figure 3.3: The effects of ADO and A1R analogs on fictive episodes per minute in intact
brainstem preparations.
Figure 3.4: The effects of ADO and A1R analogs on fictive breaths per episode in intact
brainstem preparations.
Figure 3.5: The effects of ADO and A1R analogs on fictive breath area in intact brainstem
preparations.
Figure 3.6: The effects of ADO and A1R analogs on fictive breath duration in intact brainstem
preparations.
Figure 3.7: The effects of ADO and A1R analogs on the total fictive ventilation index in intact
brainstem preparations.
xi
Figure 4.1: Overview of the stages and general procedures taken within the midbrain transection
experiment.
Figure 4.2: The effects of transecting the midbrain on fictive breathing frequency.
Figure 4.3: The effects of transecting the midbrain on fictive episodes per minute.
Figure 4.4: The effects of transecting the midbrain on fictive breaths per episode.
Figure 4.5: The effects of transecting the midbrain on fictive breath area.
Figure 4.6: The effects of transecting the midbrain on fictive breath duration.
Figure 4.7: The effects of transecting the midbrain on the total fictive ventilation index.
Figure 5.1: Overview of the stages and general procedures taken within the experiment on
midbrain transected preparations.
Figure 5.2: The effects of ADO and A1R analogs on fictive breathing frequency in midbrain
transected brainstem preparations.
Figure 5.3: The difference in effects of ADO and A1R analogs on fictive breathing frequency
between intact brainstem preparations and midbrain transected brainstem preparations during the
dose and pH treatment period.
Figure 5.4: The effects of ADO and A1R analogs on fictive episodes per minute in midbrain
transected brainstem preparations.
Figure 5.5: The difference in effects of ADO and A1R analogs on fictive episodes per minute
between intact brainstem preparations and midbrain transected brainstem preparations during the
dose and pH treatment period.
Figure 5.6: The effects of ADO and A1R analogs on fictive breaths per episode in midbrain
transected brainstem preparations.
xii
Figure 5.7: The difference in effects of ADO and A1R analogs on fictive breaths per episode
between intact brainstem preparations and midbrain transected brainstem preparations during the
dose and pH treatment period.
Figure 5.8: The effects of ADO and A1R analogs on fictive breath area in midbrain transected
brainstem preparations.
Figure 5.9: The difference in effects of ADO and A1R analogs on fictive breaths are between
intact brainstem preparations and midbrain transected brainstem preparations during the dose and
pH treatment period.
Figure 5.10: The effects of ADO and A1R analogs on fictive breath duration in midbrain
transected brainstem preparations.
Figure 5.11: The difference in effects of ADO and A1R analogs on fictive breath duration
between intact brainstem preparations and midbrain transected brainstem preparations during the
dose and pH treatment period.
Figure 5.12: The effects of ADO and A1R analogs on TFV in midbrain transected brainstem
preparations.
Figure 5.13: The difference in effects of ADO and A1R analogs on TFV between intact
brainstem preparations and midbrain transected brainstem preparations during the dose and pH
treatment period.
Figure A.1: Fictive breathing frequency (fictive breaths per minute) measured during the pre-
treatment phase of the experiment in all groups examined within the current study.
Figure A.2: Fictive episodes per minute measured during the pre-treatment phase of the
experiment in all groups examined within the current study.
Figure A.3: Fictive breaths per episode measured during the pre-treatment phase of the
experiment in all groups examined within the current study.
Figure A.4: Total fictive ventilation index (V s/min) measured during the pre-treatment phase of
the experiment in all groups examined within the current study.
xiii
Figure A.5: Integrated area of the fictive breaths measured during the pre-treatment phase of the
experiment in all groups examined within the current study.
Figure A.6: The fictive breath duration measured during the pre-treatment phase of the
experiment in all groups examined within the current study.
Figure A.7: The effect of time on respiratory variables in intact control in vitro brainstem
preparations.
Figure A.8: Fictive breathing (vagal motor output) traces from midbrain transaction
experiments.
1
Chapter 1
General Introduction
2
1.1 Breathing in Animals
In general, respiratory rhythmogenesis in all animals is produced centrally and can be modified
by central and afferent inputs. Respiratory centers and rhythm generating neurons that are found
in the medulla , such as the inspiratory (I) neurons located within the preBötzinger complex
(PreBötzC), are crucial to the constitution of the basic rhythm of breathing in mammals and other
vertebrates (Smith et al., 1991; Rekling and Feldman, 1998; Gray et al., 1999; Feldman et al.,
2003). The basic respiratory rhythm is subjected to modification by central CO2/pH
chemoreceptors and peripherial (arterial) chemoreceptors. The central CO2/pH chemoreceptors
respond to pH changes associated with changing CO2 levels within the cerebrospinal fluid and
the peripheral chemoreceptors monitor PCO2, pH and PO2 of the arterial blood. Pulmonary
stretch receptors (PSR) are mechanoreceptors found on the lung that monitor the degree of lung
inflation/deflation and can have significant modulatory effects on respiratory rhythm and
breathing pattern formation.
The morphology of the respiratory system differs amongst various vertebrate groups. The form
and function of the respiratory system is usually tailored to exploit the external environment such
that the respiratory system provides the organism with adequate oxygen, expels carbon dioxide
(CO2) to the external environment and maintains homeostasis of arterial O2 and CO2 levels
(Wasserman, 1978; Cohn, 1983; Kinkead, 1997).
1.2 Breathing in Amphibians
Breathing in amphibians is dynamic as the respiratory organs undergo morphological changes
throughout the life cycle. During pre-metamorphosis, the respiratory organs present in tadpoles
are the gills and the skin. Tadpoles are water breathers and utilize cutaneous respiration for
approximately 60% of gas exchange and the gills for 40% for both O2 uptake and CO2 excretion.
During metamorphosis, the amphibian becomes a facultative air-breather marked by the retention
of the gills and the progressive development of the lungs. The emergence of air breathing that
occurs during metamorphosis is believed to be triggered by an increase in circulating
corticosterone and concurrent changes to GABAergic neurotransmission (Fournier et al., 2012).
During post-metamorphosis, the adult amphibian switches from facultative air-breathing to
obligate air-breathing. At the adult stage, the gills have completely rescinded and the lungs
3
become the primary respiratory organ for O2 uptake (approximately 90%) with the skin still
playing a role in CO2 excretion (Burggren and West, 1982; Pinder and Burggren, 1986; Wells,
2007).
Like other animals, anuran amphibians possess a central rhythm generator (CRG) for lung
ventilation. However, unlike other animals, amphibians also have a CRG that regulates the non-
ventilatory buccal oscillations that occur between breaths (Wilson et al., 2002). Unlike mammals
that utilize a negative pressure pump to inflate and deflate the lungs, amphibians utilize a buccal
force pump (positive pressure pump) to control lung inflation (Jones, 1982).
The pulmonary respiratory system of amphibians consists of the buccal cavity and the lungs.
Inbetween the buccal cavity and the lungs is the glottis, which controls the passage of air
between the two internal spaces. The step-by-step process behind amphibian breathing has been
described by several studies (West and Jones, 1975; Macintyre and Toews, 1976; Vitalis and
Shelton, 1990; Jones, 1982). First, air enters the buccal cavity through the nares by negative
pressure generated by the lowering of the floor of the buccal cavity. Air in the lungs remains
trapped within the lungs due to the closed glottis. When the glottis opens, the pre-existing air
held in the lungs from the previous breath is drawn into the upper region of the buccal cavity,
while the newly inspired air remains in the lower portion of the buccal cavity. The expired air
from the lungs then exits through the open nares into the atmosphere. The nares will close while
the glottis remains open, and the newly inspired air gets pushed into the lungs by positive
pressure that is generated by the upward contraction of the floor of the buccal cavity. The glottis
then closes, which completes a single lung breath. Since the surface of the buccal cavity lacks
vasculature, only air in the lungs is believed to be involved with gas exchange. Lung inflation
cycles, in which the lung is “pumped” up by successive breaths, are associated with elevated
respiratory drive, defense behaviours and preparation for vocalization. Deflation cycles follow
inflation cycles after a period of apnea (Gargaglioni and Milsom, 2007).
4
Figure 1.1. The respiratory cycle of the adult anuran amphibian. The inhalation of air begins with the generation of
negative pressure in the buccal cavity (achieved by the depression of the floor in the buccal cavity) which draws in
atmospheric air through the open nares into the lower half of the buccal cavity (A). The pre-existing air in the lungs
is under high pressure and when the glottis opens the pre-existing air in the lungs gets pushed out in to the
atmosphere through the open nares (B). The nares then close and the air trapped in the lower half of the buccal
cavity get pumped into the lungs through the open glottis by the elevation of the floor of the buccal cavity (C). The
glottis closes and the nares re-open which allows the cycle to either begin again or allow buccal oscillations to occur
without lung ventilation.
1.3 Breathing Patterns in Amphibians
The pattern of breathing displayed by the adult anuran is described as intermittent (i.e.
discontinuous) (Milsom, 1991). The intermittent breathing pattern can be further described as
single breaths and/or clusters of breaths into doublets/triplets (two or three breaths in succession)
that are randomly distributed and separated by a brief period of apnea. Indeed it is possible to see
almost any type of “random” distribution of breaths in discontinuously breathing animals. In
contrast to the intermittent breath pattern displayed by amphibians, euthermic mammals, birds
and water-breathing fish all have a breathing pattern that is continuous. The stark difference
between breathing patterns observed in these animals is likely due to metabolic demands, which
are significantly lower in amphibians compared to birds and mammals (Gargaglioni and Milsom,
2007). In addition, the maintenance of the episodic breath patterns in the adult anuran amphibian
5
appears to be independent of fluctuations in blood-gas levels (West et al., 1987; Kinkead et al.,
1994, 1997; Milsom et al., 1999; Reid et al., 2000).
Under certain conditions, the breathing pattern in both mammals and amphibians can be
switched (i.e. mammals can breathe episodically and adult amphibians can breathe
continuously). In mammals, episodic breathing can occur during hibernation, when metabolic
demands are low (Kinkead et al., 1997). In amphibians, the breathing pattern can be modified by
the level of respiratory drive. During elevated respiratory drive, the frequency of breaths
increases and the apnea periods between each breath shortens. Elevated respiratory drive can
occur by external stressors, such as hypoxia (low O2 levels) or hypercapnia (high CO2 levels).
When respiratory drive becomes extremely high, especially during severe hypercapnia,
amphibians exhibit continuous breathing (Milsom, 1991; Gargaglioni and Milsom, 2007).
Evidence from past studies has suggested that the caudal half of the midbrain is involved in the
clustering of breaths into episodes (Reid et al., 2000a; McAneney and Reid, 2007).
1.4 Control of Breathing
Regulation of breathing in amphibians can be described as a complex network of interactions
between stimulatory inputs from central and arterial chemoreceptors, and inhibitory inputs from
olfactory chemoreceptors in conjunction with CO2-sensitive tonic and/or phasic pulmonary
stretch receptor (PSR) feedback (Kinkead and Milsom, 1996). The interaction between
peripheral and central chemoreceptors is seen during both the acute hypercapnic ventilatory
response and the acute hypoxic ventilatory response (West et al., 1987; Smatresk and Smits,
1991; Reid, 2006). As in other animals, central integration of the afferent feedback provided by
these receptors leads to modified motor output to the respiratory muscles.
1.4.1 Central Processes
In mammals, the basic rhythm of breathing is generated centrally in the preBötzC and the
parafacial respiratory group (pFRG), which are located within the rostro-ventral medulla
(Onimaru and Homma, 2003; Feldman and Del Negro, 2006; Wilson et al., 2006; Gargaglioni
and Milsom, 2007). Within the preBӧtzC are inspiratory (I) neurons, which fire during
inspiration (Smith et al., 1991; Rekling and Feldman, 1998; Gray et al., 1999; Feldman et al.,
2003). Within the pFRG are pre-I neurons that are coupled to the I-neurons of the preBӧtzC. The
6
pre-I neurons are believed to trigger the onset of bursting in the preBӧtzC (Smith et al., 1991;
Mellen et al., 2003; Onimaru and Homma, 2003; Feldman and Del Negro, 2006).
Regions within the medulla of anuran amphibians generates respiratory rhythm (McLean et al.,
1995a; Reid and Milsom, 1998; Reid et al., 2000a; Torgerson et al., 2001). In bullfrogs, the
ventral medullary reticular formation contains two rythmogenic sites involved with the
generation of endogenous respiratory activity (Vasilakos et al., 2005). One of these rythmogenic
sites can be found between the VIIth (facial) and IXth (glossopharyngeal) cranial nerves, which
is critical for lung ventilation. The other rythmogenic site is found at the level of the Xth (vagus)
nerve root and is required for buccal oscillation regulation (McLean et al., 1995b; Wilson et al.,
2002). During anuran development, the location of rhythm generation changes; in tadpoles,
respiratory rhythm generation occurs caudal to cranial nerve X and during metamorphosis to the
adult form, rhythm generation moves to a more rostral brainstem site (Torgerson et al., 2001).
1.4.2 Olfactory Chemoreceptors
The nasal mucosa of anuran amphibian contains CO2-sensitive olfactory chemoreceptors. These
receptors send an afferent signal to the brain via the trigeminal and olfactory nerves when
stimulated by high levels of CO2 (Sakakibara, 1978). Past studies have demonstrated that when
stimulated by CO2 the olfactory chemoreceptors produce an inhibitory signal that reduces
breathing frequency (Sakakibara, 1978; Ballam and Coates, 1989; Coates, 2001; Kinkead and
Milsom, 1996; Milsom et al., 2004). The degree of inhibition on breathing frequency from the
olfactory chemoreceptors is dependent on the level of CO2 with higher levels of CO2 leading to
greater levels of breathing frequency inhibition.
1.4.3 Pulmonary Stretch Receptors
Pulmonary stretch receptors (PSR) are mechanoreceptors that are located in the walls of the
lungs. These receptors monitor changes in lung volume during inspiration, expiration, and apnea,
and send their feedback to the brain via the pulmonary vagus nerve. PSR feedback can either
promote or inhibit respiratory activity depending on whether the PSR feedback is tonic or phasic.
Phasic PSR feedback stimulates breathing frequency via increases in the number of breathing
episodes (Reid and West, 2004). Tonic PSR feedback is considered to be inhibitory despite the
fact that it stimulates breathing frequency. This is because tonic PSR feedback, unlike phasic
7
PSR feedback, stimulates breathing frequency by promoting lung deflation breaths instead of
lung inflation breaths (Sanders and Milsom, 2001). As such, this form of PSR feedback leads to
an acceleration in lung deflation. Tonic PSR feedback can also inhibit peripheral chemoreceptor-
induced increases in breathing under conditions of minimal stimulation from central pH/CO2
chemoreceptors (Wang et al., 2004). In anuran amphibians, PSR activity is sensitive to the level
of CO2 becoming inhibited with increasing levels of CO2 (Milsom and Jones, 1977; Kuhlman
and Fedde, 1979). In addition, depending on the level of CO2, tonic PSR feedback can function
to alter breathing pattern and the form of the individual breaths (Sanders and Milsom, 2001).
PSR feedback can also interact with central pH/CO2 chemoreceptor activity and enhance the
effects of aCSF pH on respiratory frequency (Kinkead et al. 1994).
1.4.4 Peripheral O2/CO2/pH sensitive chemoreceptors
The peripheral chemoreceptors are found in the aortic bodies within the aortic arch and the
carotid bodies at the bifurcation of the common carotid arteries. These receptors monitor the
PCO2, pH and PO2 of the arterial blood and when stimulated (during hypoxia and hypercapnia),
will transmit an afferent signal through the vagus and glossopharyngeal nerves to the respiratory
centers to increase breathing (West et al., 1987; Smatresk and Smits, 1991). Although arterial
chemoreceptors in the carotid labyrinth and aortic arch are both O2 and CO2-sensitive, exposing
the peripheral chemoreceptors in the carotid labyrinth to elevated levels of O2 will reduce
discharge at any given level of CO2 (Van Vliet and West, 1992). A comparison of respiratory
recordings between intact animals and in vitro brainstem-spinal cord preparations from
amphibians indicates that without afferent feedback from peripheral chemoreceptors, less
breathing activity is observed (Kinkead et al., 1994; Reid et al., 2000b; Reid, 2006).
1.4.5 Central pH/CO2 sensitive chemoreceptors
The central chemoreceptors found on the ventrolateral surface of the medulla sense the pH
associated with the CO2 level within the cerebrospinal fluid (CSF) (Smatresk and Smits, 1991;
Milsom, 2002; Taylor et al., 2003a). These receptors directly synapse with the respiratory
centers and are stimulated by reductions in CSF pH or increases in CSF CO2 levels (Smatresk
and Smits, 1991; Milsom, 2002). Stimulation of these receptors leads to an increase in
respiratory drive.
8
1.4.6 Neurotransmitters
Several studies have examined the effects of neurotransmitters on respiration in anuran
amphibians (McLean et al., 1995a; Hedrick et al., 1998; Hendrick et al., 2005; Broch et al.,
2002; Straus et al., 2000). Neurotransmitters such as GABA and glycine have been demonstrated
to exert an inhibitory modulation to breathing frequency in amphibians (McLean et al., 1995a;
Broch et al., 2002). In addition, the clustering of breaths into episodes observed in amphibian
breathing may be regulated by the GABAB pathway (Straus et al., 2000). The neurotransmitter
that has been demonstrated to exert a stimulatory effect on breathing frequency in amphibians is
glutamate (McLean et al., 1995a). Studies by Hendrick et al. (1998 & 2005) demonstrated that
preventing NO generation via NMDA receptor (a glutamate receptor) activation results in the
abolishment of respiratory rhythm in the bullfrog (Rana catesbeiana) illustrating the importance
of NMDA receptors in respiratory rhythm generation in amphibians. Collectively, the effects of
various neurotransmitters on central respiratory modulation in amphibians are similar to those
observed in mammals, which suggests that a common mechanism to central respiratory rhythm
generation exists between the two (Wang et al., 1999).
In current literature the effects of serotonin, dopamine and noradrenaline on breathing have been
documented in mammals but not in anuran amphibians. The neurotransmitter serotonin has been
demonstrated to be a breathing stimulant in the CNS, producing increases in respiration during
normoxia (Millhorn et al., 1980). The neurotransmitter noradrenaline has a complex effect on
breathing, acting at the carotid bodies as a stimulant (Eldridge and Gill-Kunar, 1980) and as a
depressant at the CNS (Champagnat et al., 1978). The neurotransmitter dopamine also has a
complex effect on breathing, however opposite to noradrenaline, acting on the carotid body as a
ventilatory depressor and acting as a stimulant in the CNS (Nakano et al., 2002).
1.5 In vitro Brainstem – Spinal Cord Preparation
An in vitro brainstem preparation consists of the spinal cord and brainstem (which includes a
functioning medulla) as well as the midbrain. Using the in vitro brainstem preparation, fictive
breathing (motor output from respiratory nerves) can be measured from the cranial nerves.
Respiratory-related cranial nerves include the trigeminal nerves and hypoglossal nerves, which
regulate the raising or lowering of the floor of the buccal cavity, or the vagus nerve, a branch of
which controls the opening and closing of the glottis (Sakakibara, 1948a,b). The experimental
9
significance of the in vitro brainstem preparation is that it allows research to be done solely on
the central control of breathing, without any regulation or effects from peripheral input (Reid and
Milsom, 1998). In addition, in vitro brainstem preparations from amphibians can remain viable
for longer periods of time compared to mammals (some for more than 20 hours) due to their
lower metabolic rates and enhanced hypoxia tolerance (Morralles and Hedrick, 2002; Reid and
Milsom, 1998).
1.6 Adenosine
ADO is a neuromodulator / neurotransmitter that is generated by all living cells during the
breakdown of adenosine monophosphate (AMP) by ecto-5’-nucleotidase or cytosolic-5’-
nucleotidase (Latini and Pedata, 2001). When the intracellular concentration of ADO becomes
greater than that of the extracellular fluid, ADO is transported out of the cell and into the
extracellular fluid via nucleoside transporters. In the extracellular space, ADO can interact with
four different subgroups of ADO receptors (A1, A2A, A2B and A3), each of which have been
classified based on ligand affinities, structure, function and molecule interaction (Dohrman et al.,
1997). With reference to mammalian models, the A1 receptor (A1R) has the highest affinity for
ADO and is also the most abundant ADO receptor in the brain with the highest expression in the
cortex, cerebellum, hippocampus, and dorsal horn of the spinal cord (Goodman and Snyder,
1982; Dixon et al., 1996; Ribeiro et al., 2003). Coupled to the A1R are the inhibitory G proteins
(Gi) that function to hyperpolarize cells and reduce neuronal excitability when activated.
The A2AR has the second highest affinity for ADO and is also the second most abundant ADO
receptor in the brain that is found highly concentrated in the striatum and olfactory bulbs. Unlike
the A1R, the A2AR is coupled to stimulatory G proteins (Gs) and when activated will promote
cellular depolarization. Much like the A2AR, the A2BR is also stimulatory, but is less abundant
and has a lower affinity for ADO. Lastly the A3R, which functions similarly to the A1R, is
considered to be the least expressed adenosinergic receptor in the CNS with the least affinity for
ADO amongst the adenosinergic receptor subtypes (Ciruela et al., 2010).
The A1Rs and the A2ARs are primarily responsible for the central effects of ADO (Dunwiddie,
and Masino, 2001). The primary mode of effect of the A1R and the A2AR is the mediation of the
secondary-messenger enzyme adenylate cyclase (AC). Stimulation of the A1R activates the Gi
coupled proteins, which inhibit the enzyme AC and subsequent production of cAMP (Klinger et
10
al., 2002). On the contrary, stimulation of the A2AR activates the Gs coupled proteins, which
stimulate the AC and subsequent production of cAMP. The activation of the A1R and A2AR also
causes changes to ion channels. Activation of presynaptic A1Rs directly inhibits voltage-
dependent calcium (Ca2+
) channels (Ciruela et al., 2010). In addition, A1R activation produces
hyperpolarisation in a manner independent of cAMP by inducing a potassium (K+) current via
GIRK (G protein-gated inwardly rectifying K+ channels) (Klinger et al., 2002). In contrast, the
activation of A2ARs results in a Ca2+
-dependent release of glutamate and acetylcholine, by means
of a mechanism that may involve P-type Ca2+
channels (Ciruela et al., 2010).
1.7 Hypothesis and Objectives
A previous study from this lab investigated the effects of chronic hypoxia (CH) on central
respiratory-related pH/CO2 chemosensitivity in cane toads (Bufo marinus) (McAneney and Reid,
2007). Using in vitro brainstem–spinal cord preparations from cane toads, McAneney and Reid
(2007) had demonstrated that under normal physiological conditions, descending inputs from
the midbrain function to inhibit or reduce fictive breathing frequency during elevated respiratory
drive (i.e., during exposure of the preparations to lower levels of aCSF pH). In addition to the
study, the authors demonstrated that when cane toads were exposed to CH the inhibitory
modulation from these descending inputs from the midbrain became strengthened (i.e. fictive
breathing frequency was reduced even further).
The aim of the current study was to examine the mechanisms exerted by these descending
midbrain inputs that contribute to the inhibitory modulation of breathing that was observed in the
previous study. Since ADO is a naturally circulating metabolite that has been demonstrated to
increase in the extracellular fluid during CH (Klinger et al., 2002; Pamenter et al., 2008; Latini
and Pedata, 2001), I hypothesized that these descending inputs from the midbrain suppresses
respiratory activity through purinergic regulation by ADO acting primarily through the A1R. In
current literature, the effect of extracellular ADO on in vitro respiratory activity of the cane toad
has not been documented. As such, my primary objectives include the examination of the acute
effects of extracellular ADO on fictive breathing measured from the isolated brainstem-spinal
cord preparation and the isolation of the primary ADO receptor that is acted upon by ADO to
exert these effects.
11
To test the hypothesis, I superfused ADO, the A1R antagonist 8-Cyclopentyl-1,3-
dipropylxanthine (DPCPX) and the A1R agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA),
onto in vitro brainstem-spinal cord preparations while recording fictive breathing. To isolate the
effects of ADO and the A1R analogs, on fictive breathing, from descending midbrain inputs, the
superfusion of ADO and A1R analogs was repeated on midbrain transected in vitro brainstem
preparations.
12
Chapter 2
General Materials and Methods
13
2.1 Experimental Animals
Cane toads were obtained from a commercial supplier (Boreal Scientific, St Catherine’s, Ontario,
Canada). They were housed in the aquatics facility at the University of Toronto Scarborough.
Toads were kept at room temperature (20-22ºC) and held in fibreglass tanks that contained
plastic tubes and trays filled with de-chlorinated water to mimic both terrestrial and aquatic
habitats. The photoperiod was maintained at 12 h light; 12 h dark. The toads were fed once a
week with either live meal worms or crickets. All procedures were approved by the University of
Toronto Animal Care Committee and conform to standards set by the Canadian Council for
Animal Care.
2.2 Artificial Cerebral Spinal Fluid (aCSF) Solution
The artificial cerebral spinal fluid (aCSF) used to superfuse the isolated brainstem-spinal cord
preparations contained 103.5 mM NaCl, 25 mM NaHCO3, 4 mM KCl, 10 mM D-glucose, 1.36
mM MgCl2 and 2.43 mM CaCl2(all chemicals from Sigma) (Taylor et al., 2003a,b; Gheshmy et
al., 2006). The aCSF used for the surgical removal of the brainstem-spinal cord was placed in an
ice bath and gassed with 100% O2. The aCSF used during the experimental protocol was gassed
with both CO2 and O2 to achieve an aCSF pH between 7.4 and 8.0. All gas flow levels were
recorded using a gas flow meter (Smart-Trak 2000, Sierra instruments INC.). The pH of the
aCSF was measured constantly with a pH meter (VWR) submerged in the aCSF reservoir. It
was calibrated using standard buffer solutions.
14
Figure 2.1: Schematic diagram of the apparatus used to measure fictive breathing (respiratory motor output) from
isolated cane toad brainstem-spinal cord preparations. (a) aCSF reservoir (b) pH meter (c) CO2 inflow (d) O2 inflow
(e) Peristaltic pumps to circulate the aCSF (f) Preparation chamber (g) Suction electrode (h) Amplifiers and
integrator (i) data acquisition system.
2.3 The In vitro Brainstem-Spinal Cord Preparation
Toads were anaesthetised by submersion in a solution containing 500 ml of cold water mixed
with 0.5g of 3-aminobenzoic acid ethyl ester (MS222, 1.0g 1-1
; Sigma) and 1.0g of sodium
bicarbonate to buffer the solution to pH 7.0 (Reid and Milsom,1998; Reid et al., 2000a; Reid et
al., 2000b; Gheshmy et al., 2006). Once in the anaesthetic, toads were monitored for cessation of
buccal movements as well as eye-blink and toe-pinch reflexes. When these reflexes were
eliminated, the surgical removal of the brainstem-spinal cord commenced. An incision was
made in the skull rostral to the optic lobes and along the sides of the spinal cord. Using the bone
shears, the spinal cord was severed at the level of the third spinal nerve. The cranial case as well
as the associated dermal tissues and muscles surrounding the spinal cord were removed with
rongeurs and bone shears. Using Westcott scissors, the olfactory bulb was removed (Gheshmy et
al., 2007) and the cranial nerves were cut close to their exit from the skull. The brain preparation
was then isolated from the brain case and transferred to and immobilized within a Sylgard-coated
dissecting dish. With the aid of a light microscope, the dura mater surrounding the brain was
removed with tweezers to free the cranial nerve roots. Throughout the surgical procedure, a
15
constant supply of cold, oxygenated aCSF was applied to keep the isolated brain preparation
viable. The brainstem preparation was then transferred to the preparation recording chamber
(Fig. 2.1) and immobilized with insect pins. The trigeminal nerve root was aspirated into a
suction electrode and the preparation was kept in the recording chamber and superfused with the
aCSF solution (pH of 7.7-7.8) until respiratory motor output was stabile (approximately one
hour).
2.4 Experimental protocol: aCSF pH Changes and Treatment
with Adenosine, CCPA or DPCPX
Once the motor output from the isolated brainstem-spinal cord preparation had stabilised (i.e.
rhythmic recording became consistently visible), respiratory motor activity was recorded for data
analysis purposes for the final 10 minutes of that period (i.e., the stabilisation period). Following
the stabilisation period, the pH of the aCSF was changed to either 7.4, 7.6, 7.8 or 8.0. For each
isolated brain preparation the sequence of pH changes was randomized. The aCSF was
maintained at a given pH level for 15 minutes at which point the pH was changed again such that
the preparation was exposed to all four different aCSF pH levels. The motor output (fictive
breathing) during the last 10 minutes of exposure to each pH level was recorded for data analysis
purposes. After exposing the preparation to the various aCSF pH changes, the pH was readjusted
back to 7.8. At this point the aCSF was changed such that the new aCSF superfusing the
preparation contained either 1) 1 μM adenosine (n = 9), 2) 10 μM adenosine (n = 9), 3) 10 µM
of the A1R antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; n = 9) 4) 10 µM of the A1R
agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; n = 9) or 5) a control solution of normal
aCSF (n = 8). The new aCSF solution with either adenosine, DPCPA or CCPA was gassed with
O2 and CO2 to achieve a pH level between 7.7-7.8 and the preparation was superfused for a 40
minute stabilisation period. The last 10 minutes of the stabilisation period was used for data
analysis. The current study analysed the data after 30 minutes of drug exposure to ensure
maximum dose efficiency (Pamenter et al., 2008). After the 40 minute stabilisation period, the
aCSF pH was changed to either to 7.4, 7.8, 7.6 and 8.0 as described above in the continued
presence of adenosine, CCPA or DPCPX (aka dose & pH treatment period). Each pH change
lasted 15 minutes and motor output was recorded in the last 10 minutes of each pH change for
data analysis. Figure 2.2 summarizes the experimental procedure used in this type of
16
experiments. The data for this specific series of experiments (pH changes followed by drug
treatment followed by pH changes) is reported in chapter 3. Modifications to this protocol are
reported in subsequent chapters, as appropriate.
Figure 2.2: Flow chart illustrating the general stages of the experimental protocol used in the various series of
experiments outlined in this thesis. The first step was the stabilisation period, where the brainstem preparation was
placed into a bath of circulating aCSF at constant pH 7.8 until breathing becomes rhythmic for at least 15 minutes.
The second step is the pH treatment period, where the brainstem preparation was subjected to random alterations of
aCSF pH (7.4, 7.6, 7.8 and 8.0). The third step was the experimental manipulation period in which a dose of
adenosine, CCPA or DPCPX was introduced into the aCSF superfusing the brainstem preparations that were either
kept intact or transected at the midbrain. The fourth step was the “dose and pH treatment period”, where the
brainstem preparation was exposed to random alterations in aCSF in the continued presence of adenosine, CCPA or
DPCPX.
17
2.5 Experimental protocol: Midbrain Transection
These experiments used a similar procedure as described above. However, in addition to the
aCSF solution containing adenosine or an A1R agonist or antagonist, the midbrain was removed
from the isolated brainstem-spinal cord preparations. A transection through the optic lobes on the
dorsal surface to the caudal end of the acuate periventricular nucleus on the ventral surface was
made to remove the midbrain. Based on the Hoffmann (1973) atlas of the toad brain, this
transection site would correspond to 3.5 mm posterior to the zero mark (Fig. 2.3). The
transection is rostral to the nucleus isthmi (see Figs. 29 and 30 from Hoffmann, 1973). Further
details on the midbrain transection protocols are provided in chapters 4 and 5.
Figure 2.3: A lateral view of the toad brain illustrating the transected areas (dotted lines) used in the current study.
The rostral- and caudal-most transections were used to generate the in vitro brainstem preparation. The mid-brain
transection through the optic lobes was an experimental manipulation outlined in chapters 4 and 5. NI indicates the
location of the nucleus isthmi. V, trigeminal nerve root; VII, facial nerve root; IX, glossopharyngeal nerve root; X,
vagus nerve root.
18
2.6 Controls
In experiments conducted on intact preparations, control groups were intact brainstem
preparations exposed to aCSF that did not contain adenosine, CCPA or DPCPX. Similarly,
intact brainstem preparations exposed only to aCSF (no pharmacological treatment) were used as
controls for experiments examining the effects of the midbrain transection only (prior to drug
treatment). In experiments conducted on midbrain transected preparations, control groups were
midbrain transected preparations that were exposed to virgin aCSF during the dose stabilisation
and the dose & pH treatment period of the experiment. The pH level of 7.8 was used as the
control pH level during both pH treatment periods in the pre-treatment and post-treatment phase
of the experiment.
2.7 Data and statistical analyses
AcqKnowledge 3.7.3 (Biopac Systems) software was used to acquire and store the nerve
recordings from the in vitro brainstem preparations. The data recorded from the final 10 minutes
of each experimental period and/or aCSF pH level was used for analysis. Using the criteria
described by Reid and Milsom (1998), the brain traces were analysed to determine fictive
breathing frequency (fictive breaths/min), the number of fictive breathing episodes per min, the
number of fictive breaths per episode, fictive breath duration, and the integrated area of the
fictive breaths. Fictive breaths in a given episode were defined as occurring within 2 seconds of
each other according to general practices in the literature (Kinkead et al., 1994; Kinkead et al.,
1997; Reid et al., 2000a; Gheshmy et al., 2006). The product of integrated area of fictive breaths
and fictive breathing frequency yields the total fictive ventilation (mV•sec/min). The
relationships between the respiratory variables are illustrated in Fig. 2.3. To maintain analytical
consistency amongst all experimental groups, only neural respiratory recordings that had a mean
average in pre-treatment values that were approximately equal to that observed in controls were
used in the experiment (see Appendix). All statistical analyses were performed using commercial
software (SigmaStat 3.0, SPSS). See chapters 3-5 for further details on data and statistical
analysis appropriate to the given experiments.
19
Figure 2.4: Pyramid illustrating the relationship between respiratory variables evaluated in the current study. The
total fictive ventilation index is the product of fictive breathing frequency and breath area. Fictive breathing
frequency is the product of fictive episodes per minute and fictive breaths per episode. Fictive breath duration and
amplitude are components that make up fictive breath area.
20
Chapter 3
The Effects of Adenosine, CCPA and DPCPX on intact Brainstem-Spinal Cord Preparations
21
3.1 Introduction
In general, anuran amphibians are vulnerable to dehydration and lethal heat due to the high
permeability of their skin. However, despite this vulnerability, many amphibians can be found in
diverse habitats some of which have extreme climates, such as the dry and extreme heat of
deserts or the frigid cold temperatures of northern temperate climes (Boutilier et al., 1997). To
survive such extreme environmental conditions, amphibians utilize avoidance strategies such as
burrowing underground or overwintering under water. However, inhabiting underground
burrows or submergence under water introduces another problem for amphibians as both habitats
lack sufficient oxygen and may also, at least underground, have elevated levels of carbon dioxide
(Boggs et al., 1984; Schaefer and Sadleir, 1979). However, it has been documented that
amphibians can remain within these environments for several weeks or months (Breckenridge
and Tester, 1961; Pinder et al., 1992). Furthermore, in comparison to mammals, amphibians
have a greater tolerance to hypoxia (Boutilier, 2001) and can survive conditions of persistent
hypoxia by undergoing physiological adjustments to respiratory gas exchange and metabolism.
Inside underground burrows or underwater, amphibians may enter a state of torpor (i.e.,
aestivation). In this state, there is a reduction in basal metabolic rate and metabolic demand
which is vital to surviving prolonged periods in hypoxic environments. A reduction in metabolic
activity is usually observed in amphibians during exposure to high temperatures and low water
availability (Pinder et al., 1992) and as well as during exposure to cold temperatures (Lemckert,
2004; Yu and Guo, 2010; Bickler and Buck, 2007). The advantages of metabolic depression are
that it decreases the consumption of limited resources, such as oxygen and water (Boutilier,
2001), it allows for low levels of oxidative phosphorylation to meet ATP demand (Bickler and
Buck, 2007) and it lessens the impact of ATP demand on endogenous energy reserves
(Hochachka and Guppy, 1987; Storey and Storey, 1990; Flanigan et al.1991; Guppy et al.1994).
In addition to metabolic changes, respiratory activity in amphibians changes during hypoxia.
During exposure to moderate levels of acute hypoxia (10-15% O2), amphibians generally
increase their breathing in order to maintain oxygen homeostasis (Burggren and Doyle, 1987;
Kruhøffer et al., 1987; Smatresk and Smits, 1991). However, when hypoxic exposure becomes
chronic, some amphibians have been shown to exhibit a depression in respiratory activity
(McAneney et al., 2006; McAneney and Reid, 2007). The reduction in respiratory activity
22
observed during chronic hypoxia (CH) may serve as a means of conserving energy, since the
reduction in neuronal activity regulating breathing would help to reduce brain ATP demand
(Buck and Bickler, 2007) and motor activity of respiratory muscles would be reduced.
During exposure to hypoxia, brain ATP levels decline progressively (Winmill et al., 2005), while
the extracellular levels of potassium (K+) (Winmill et al., 2005) and adenosine (ADO) increase
progressively (Klinger et al., 2002; Pamenter et al., 2008; Latini and Pedata, 2001). Multiple
hypoxic/anoxic studies suggest that adenosine enhances cytoprotective mechanisms, including
the down-regulation of NMDA (N-methyl-D-aspartate) receptor activity which, in the absence of
down-regulation, is associated with excitotoxic cell death (ECD) during hypoxic and anoxic
insults (Bickler, 2004; Buck, 2004; Downey et al., 2007; Pagonopoulou et al., 2006). Other
benefits that adenosine has during hypoxia include (a) the stimulation of glycogenolysis which
provides the substrate for anaerobic glycolysis, (b) the stimulation of anaerobic glycolysis which
increases ATP production to meet demand and (c) reduction of neuronal energy requirements by
decreasing neuronal excitability (postsynaptic inhibition) as well as neurotransmitter release
(presynaptic inhibiton) (Bickler and Buck, 2007). Collectively, I suspect that adenosine maybe
the metabolite that induces the ventilatory depression exhibited by the cane toad during chronic
hypoxia (CH).
3.2 Hypothesis & Objectives
Cane toads exhibit ventilatory depression during CH (when extracellular adenosine levels are
significantly greater than during normoxic periods). As such, I hypothesized that superfusing
adenosine onto the in vitro brainstem preparation of the cane toad would cause a depression in
respiratory motor output (i.e. fictive breathing, the neural equivalent of breathing). I also
hypothesized that adenosine would work primarily via the A1R since the A1R has been
documented to be the most abundant and widely-distributed adenosinergic receptor in the brain
and that it has the highest affinity of all purinergic receptors for adenosine. In addition, A1R
stimulation leads to the release of K+ from neurons into the extracellular space which, in part,
leads to the hyperpolarization of that neuron. To determine whether a reduction in fictive
breathing is caused by adenosine and A1R stimulation, I superfused the in vitro brainstem cord
preparation with 1 μM adenosine (n = 9), 10 μM adenosine (n = 9), the A1R antagonist, 8-
Cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 µM; n = 9) or the A1R agonist, 2-chloro-N(6)-
23
cyclopentyladenosine (CCPA; 10 µM; n = 9). These pharmacological treatments were done at
various aCSF pH levels. I predicted that adenosine and CCPA treatment would reduce fictive
breathing and that DPCPX treatment would enhance fictive breathing. Furthermore, based on
previous studies I also predicted that the effects would be more pronounced at lower aCSF pH
levels.
24
3.3 Materials and Methods
See chapter 2 for details on the generation of the in vitro brainstem-spinal cord preparation and
general details of the experimental protocol for altering aCSF pH and superfusing the various
pharmacological agents onto the preparation.
Figure 3.1 illustrates the experimental protocol used in the experiments outlined in this chapter.
Briefly, the brainstem-spinal cord preparation (with the midbrain attached) was removed from
the animal and allowed to stabilise for approximately 1 hour at an aCSF pH of 7.8 (pre-treatment
stabilisation period). After this period, the aCSF pH was changed to pH levels of 8.0, 7.8, 7.6 and
7.4 (selected randomly; “pH treatment” portion of the protocol). Following these pH changes, the
preparation was superfused with aCSF containing either adenosine (1 µM; n = 9), adenosine (10
µM; n = 9), CCPA (A1R agonist; 10 µM; n = 9), DPCPX (A1R antagonist; 10 µM; n = 9), or
normal aCSF (controls; n = 8). After a stabilisation period of 40 min (“dose stabilisation” portion
of the protocol), the aCSF pH changes were repeated (“dose and pH treatment” portion of the
protocol).
25
Figure 3.1: Overview of the stages and general procedures performed within the experiments on brainstem-spinal
cord preparations reported in this chapter. The experiment was one continual process consisting of two phases; (1)
the pre-treatment phase in which the brainstem preparations were exposed to normal aCSF (no pharmacological
agents) of varying pH levels and (2) the post-treatment phase in which the brainstem preparations were exposed to a
pharmacological agent (adenosine; CCPA or DPCPX) within the aCSF as well as varying aCSF pH levels. The pre-
treatment phase of the experiment was divided into two parts; (1) the stabilisation period in which the preparation
was superfused with aCSF at a constant pH of 7.8 and (2) the pH treatment period in which the brainstem
preparation was subjected to random alterations in pH (15 minute duration for each pH change) in the aCSF. In all
cases the data were analysed in the final 10 minutes of each pH change period. The post-treatment phase of the
experiment was also split into two parts; (1) the dose stabilisation period in which the preparation was exposed to a
new aCSF reservoir containing a dose of adenosine, CCPA or DPCPX (kept at constant pH 7.8) for 40 minutes with
the data analyzed in the final 10 minutes and (2) the dose and pH treatment period in which the dose of the
pharmacological agent was still present and the brainstem preparation was subjected to random alterations to pH (15
minute duration for each pH change) in the aCSF with the data analyzed in the final 10 minutes of each pH change.
26
3.4 Data Analysis
A one-way repeated measures analysis of variance followed by a Holm-Sidak multiple
comparison test (MCT) was used to analyze the effect of altering aCSF pH levels within each
treatment group (i.e., control, adenosine, CCPA or DPCPX); the values obtained for each
respiratory variable at pH 7.4, 7.6 and 8.0 were compared to the value of the respective
respiratory variable obtained at the initial pH of 7.8 (during the stabilisation period). A one-way
analysis of variance followed by a Holm-Sidak MCT was also used to evaluate the effects of the
pharmacological agents (adenosine, CCPA or DPCPX) within the first forty minutes at constant
pH 7.8 (dose stabilisation period) by comparing the value of the respective variables between
the control group (no dose) to a specific pharmacological treatment group. To evaluate the effect
of the pharmacological treatment and aCSF pH, a two-way ANOVA with a Holm-Sidak MCT
was used. This two-way ANOVA compared the values of the respiratory variable obtained at pH
7.4, 7.6, 7.8 and 8.0 in control groups to the values of the respective respiratory variable and
relative pH set in a specific treatment group. Only post-treatment data is reported in this chapter.
The pre-treatment data are reported in the appendix. The limit of significance was 5% (p < 0.05).
The data are expressed as the mean ± one standard error of the mean (S.E.M).
27
3.5 Results
3.5.1 Fictive Breathing Frequency
Figure 3.2: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist
(DPCPX) on fictive breathing frequency measured from intact brainstem-spinal cord preparations. Panel A
illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the introduction of
the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various pH changes in
the presence of the various pharmacological agents. In both panels a number sign (#) indicates a significant
difference between the control value (aCSF with no pharmacological agents) and the values recorded with the
various pharmacological manipulations, and an asterisk sign (*) indicates a significant difference between the value
at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).
28
During the last 10 minutes of the stabilisation period (aCSF pH of 7.8 following the application
of the pharmacological agents), 1 µM adenosine (p<0.001), 10 µM adenosine (p<0.001) and
CCPA (p=0.007) caused a reduction in fictive breathing frequency while DPCPX (p<0.001)
caused a significant increase in fictive breathing frequency (Figure 3.2A).
Following the stabilisation period, the aCSF pH was changed from 7.8 to 7.4, 7.6, 7.8 or 8.0. In
the control group (Fig. 3.2B), fictive breathing frequency was increased at aCSF pH 7.4 (p =
0.001) and reduced at aCSF pH 8.0 (p = 0.004) compared to the values recorded at pH 7.8
(during the stabilisation period). In the 1 µM adenosine (p=0.018) and 10 µM adenosine (p <
0.001) groups, fictive breathing frequency increased at an aCSF pH of 7.4 compared to the value
at 7.8 during the stabilisation period. In the CCPA superfused group, fictive breathing frequency
was significantly different at pH 7.4 (p<0.001) and 7.6 (p<0.001) from the values recorded at pH
7.8. In the DPCPX superfused group, fictive breathing frequency was significantly different at
pH 8.0 (p = 0.049) from the values recorded at pH 7.8.
The reduction in fictive breathing frequency observed in response to adenosine (1 and 10 μM)
and CCPA, compared to the value in the control group, during the stabilisation period (Fig.
3.2A) was maintained during the aCSF pH changes at all pH levels with the exception of 8.0.
The increase in fictive breathing frequency observed in Fig. 3.2A with DPCPX treatment was
maintained at all aCSF pH levels (Fig. 3.2B).
29
3.5.2 Fictive Episodes per Minute
Figure 3.3: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist
(DPCPX) on the number of fictive breathing episodes per minute measured from intact brainstem-spinal cord
preparations. Panel A illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8
following the introduction of the pharmacological agents into the aCSF. Panel B illustrates the data recorded
following the various pH changes in the presence of the various pharmacological agents. In both panels, a number
sign (#) indicates a significant difference between the control value (aCSF with no pharmacological agents) and the
values recorded with the various pharmacological manipulations, and an asterisk sign (*) indicates a significant
difference between the value at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation
period (A).
30
During the stabilisation period at an aCSF pH of 7.8, the number of fictive breathing episodes
per minute was reduced, compared to the control group, in response to superfusion with 1 μM
adenosine (p = 0.005), 10 μM adenosine (p = 0.001) and CCPA (p = 0.003) while treatment with
DPCPX caused an increase in the number of fictive episodes per minute (p < 0.001) (Fig. 3.3A).
In the control group (Fig. 3.3B) lowering the aCSF pH to 7.4 (<0.001) and 7.6 (p = 0.036)
caused an increase in the number of fictive episodes per minute while raising pH to 8.0 caused a
reduction in episodes per minute (p = 0.001). In preparations superfused with adenosine (1 µM, p
= 0.006; 10 µM, p = 0.008) or CCPA (p = 0.040), reducing the aCSF pH to 7.4 caused an
increase in the number of fictive episodes per minute (Fig. 3.3B). There was no effect of altering
aCSF pH on the number of episodes per minute in the group superfused with DPCPX (p >
0.221).
During the aCSF-altering phase of the experiment (Fig. 3.3B), treatment with both doses of
adenosine and the single dose of CCPA caused a reduction in the number of fictive breathing
episodes per minute at pH levels of 7.4, 7.6 and 7.8 but not 8.0. Treatment with DPCPX caused
an increase in the number of fictive episodes per minute at pH levels of 7.4 and 7.6.
31
3.5.3 Fictive Breaths per Episode
Figure 3.4: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist
(DPCPX) on the number of fictive breaths per episode measured from intact brainstem-spinal cord preparations.
Panel A illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the
introduction of the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various
pH changes in the presence of the various pharmacological agents. In both panels, a number sign (#) indicates a
significant difference between the control value (aCSF with no pharmacological agents) and the values recorded
with the various pharmacological manipulations, and an asterisk sign (*) indicates a significant difference between
the value at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).
32
During the stabilisation period, the number of fictive breaths per episode was increased,
compared to the control group, in response to treatment with 10 µM adenosine (Fig. 3.4A; p =
0.025). The other treatments had no effect.
When compared to the values recorded at pH 7.8 in the stabilisation period, altering aCSF pH
had no effect on the number of fictive breaths per episode with the exception of the CCPA
superfused groups at pH 7.6 (Fig. 3.4B; p = 0.016).
Superfusion with 10 µM adenosine and CCPA caused an increase in the number of fictive
breaths per episode during the aCSF pH-altering phase of the experiment at aCSF pH levels of
7.4, 7.6 and 7.8 but not 8.0. Superfusion with 1 µM adenosine and DPCPX had no effect on the
number of fictive breaths per episode at any aCSF pH level.
33
3.5.4 Integrated Area of Fictive Breaths
Figure 3.5: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist
(DPCPX) on fictive breath area (mV•sec) measured from intact brainstem-spinal cord preparations. Panel A
illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the introduction of
the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various pH changes in
the presence of the various pharmacological agents. A number sign (#) indicates a significant difference between the
control value (aCSF with no pharmacological agents) and the values recorded with the various pharmacological
manipulations, and an asterisk sign (*) indicates a significant difference between the value at that particular pH
(panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).
34
During the stabilisation period at an aCSF pH of 7.8, treatment with DPCPX caused a reduction
in fictive breath area compared to the control values (Fig. 3.5A; p = 0.004). However, this
difference did not manifest during the aCSF-altering phase of the experiment (Fig. 3.5B). Neither
changing aCSF pH nor treatment with adenosine or CCPA had any effect on fictive breath area
during this phase of the experiment (Fig. 3.5B) although there were statistically significant
increases in area with DPCPX compared to the value recorded at pH 7.8 in the stabilisation
period.
35
3.5.5 Fictive Breath Duration
Figure 3.6: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist
(DPCPX) on fictive breath duration (sec) measured from intact brainstem-spinal cord preparations. Panel A
illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the introduction of
the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various pH changes in
the presence of the various pharmacological agents. In both panels a number sign (#) indicates a significant
difference between the control value (aCSF with no pharmacological agents) and the values recorded with the
various pharmacological manipulations, and an asterisk sign (*) indicates a significant difference between the value
at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).
36
During the stabilisation period at an aCSF pH of 7.8 (Fig. 3.6A), 10 µM adenosine caused a
small decrease in the duration of the fictive breaths compared to the control values (p = 0.007).
However, this decrease (at pH 7.8) was not observed during the aCSF-altering phase of the
experiment (Fig. 3.6B; p = 0.426). During the stabilisation period at an aCSF pH of 7.8 (Fig.
3.6A), there was no effect of DPCPX on fictive breath duration (p = 0.335). However, during the
pH-altering phase of the experiment there was a reduction in fictive breath duration at all aCSF
pH levels (Fig. 3.6B) compared to the control group. Neither 1 µM adenosine nor CCPA affected
fictive breath duration during any stage of the experiment.
37
3.5.6 Total Fictive Ventilation
Figure 3.7: The effects of adenosine (1 and 10 µM), an A1R agonist (CCPA, 10 µM) and an A1R antagonist
(DPCPX) on total fictive ventilation (mV•sec/min) measured from intact brainstem-spinal cord preparations. Panel
A illustrates data recorded from the 40 minute stabilisation period at an aCSF pH of 7.8 following the introduction
of the pharmacological agents into the aCSF. Panel B illustrates the data recorded following the various pH changes
in the presence of the various pharmacological agents. In both panels a number sign (#) indicates a significant
difference between the control value (aCSF with no pharmacological agents) and the values recorded with the
various pharmacological manipulations, and an asterisk sign (*) indicates a significant difference between the value
at that particular pH (panel B only) to the value recorded at pH 7.8 in the stabilisation period (A).
38
Total fictive ventilation index (TFV) is the product of the integrated fictive breath area and
fictive breathing frequency and can be considered to be the primary index of fictive breathing.
During the stabilisation period (Fig. 3.7A), treatment with 1 µM adenosine (p<0.001), 10 µM
adenosine (p<0.001) and CCPA (p=0.018) caused a reduction in total fictive ventilation. There
was no effect of DPCPX (p=0.586)
During the aCSF pH-altering phase of the experiment, in the control group there was an increase
in TFV at an aCSF pH of 7.4 (p = 0.002) and a significant decrease at an aCSF pH of 8.0 (p =
0.001) compared to the values observed at pH 7.8 during the stabilisation period. In the group
treated with 10 µM adenosine (p = 0.002) or DPCPX (p = 0.032), there was an increase in TFV
at pH 7.4 compared to the value at 7.8 while in the CCPA-treated group there were increases at
pH 7.4 and 7.6 (p<0.001) compared to the values at 7.8 (Fig. 3.7B). However there were no
significant differences observed in the TFV value between the stabilisation period and the aCSF-
altering phase in the group superfused with adenosine 1 µM (Fig. 3.7B).
During the aCSF-altering phase of the experiment, TFV was reduced at aCSF pH levels of 7.4,
7.6 and 7.8 in the adenosine (1 and 10 µM) treated groups and the CCPA-treated group; there
was no effect at pH 8.0. In contrast, TFV was augmented at aCSF pH level 8.0 in the DPCPX-
treated group but remained unaffected at aCSF pH levels of 7.4, 7.6 and 7.8 (Fig. 3.7B).
39
3.6 Discussion
The major results of the experiments outlined in this chapter are: 1) Adenosine (both doses) and
the adenosine A1R agonist, CCPA caused a reduction in total fictive ventilation (TFV) during the
stabilisation period immediately prior to changing the aCSF pH while the adenosine A1R
antagonist, DPCPX had no effect. 2) Adenosine and CCPA caused a reduction in TFV during the
aCSF-altering phase of the experiment such that TFV was reduced at pH levels 7.4 to 7.8 but not
8.0; DPCPX had no effect. 3) The reducing effects of adenosine and CCPA on TFV were
mediated by changes in fictive breathing frequency. 4) DPCPX caused an increase in fictive
breathing frequency but, given simultaneous reductions, or no changes in, fictive breath area, this
increase in fictive breathing frequency did not translate into an increase in TFV. 5) The changes
in fictive breathing frequency caused by adenosine, CCPA and DPCPX were caused by changes
in the number of fictive breathing episodes per minute. 6) The changes in the number of fictive
breaths per episode were more ambiguous and did not appear to contribute to the reduction in
fictive breathing frequency.
Given the results of this chapter, the data suggest that adenosine acting via an A1R receptor,
leads to a decrease in fictive breathing mediated predominately by changes in fictive breathing
frequency. As such, the data suggest that increases in extracellular adenosine levels in the brain
during periods of chronic hypoxia may contribute to the reduction in breathing observed in
response to chronic exposure to low inspired oxygen levels.
The activation of the A1R stimulates the opening of K+ channels and the inhibition of calcium
(Ca2+
) channels on pre-synaptic neurons, such as those that are innervated by central pH/CO2
chemoreceptor cells (Dohrman et al, 1997). Under typical conditions, CO2 in the cerebrospinal
fluid (CSF) enters into the chemoreceptor cells and become hydrated to form a proton and a
bicarbonate ion by the enzyme carbonic anhydrase. The proton and bicarbonate ions are involved
with a series of ion exchange processes, which lead to an increase in Ca2+
uptake into the cell
causing cellular depolarization and subsequent release of neurotransmitters from pre-synaptic
neurons (Lahiri and Forster, 2003). However, upon subsequent activation of A1Rs by ADO,
neuronal K+ channels open via phosphorylation by protein kinase C (PKC). In addition, A1R
activation induces a K+ current via GIRK (Klinger et al., 2002). As a result, K
+ leaves the cell
which hyperpolarizes the cell membrane. In addition, Ca2+
uptake in neuronal cells is prevented
40
by A1R mediated inhibition of neuronal Ca2+
channels via the inhibition of adenylyl cyclase
(AC), making the post-synaptic nerve less excitable and pre-synaptic nerves less likely to release
neurotransmitters. Hence an overall attenuation of breathing was observed when the brainstem-
spinal cord preparations were exposed to ADO and CCPA treatment.
The activity of the A1R also appears to be dependent on pH, as the most significant effects on
pH-sensitive fictive breathing (breathing frequency, TFV and episodes per minute) of ADO and
CCPA treatment were observed from normocapnic to hypercapnic pH levels (aCSF pH 7.8 to
7.4) and not from hypocapnic pH levels (pH 8.0). This observation is consistent with the pH-
sensitive nature of the effects of CH observed from the previous study by McAneney and Reid
(2007), suggesting that both CH and ADO do not alter pH-sensitive fictive breathing under
hypocapnic conditions but do affect pH-sensitive fictive breathing during the in vitro equivalent
of normocapnia and during an elevation in the CO2-mediated drive to breathe. Also consistent
with the study by McAneney and Reid (2007) is the fact that ADO attenuated TFV, fictive
breathing frequency and fictive episodes per minute, but had no affect on fictive breath duration
and area as observed in CH exposed toads. Collectively, the similarities between the effects of
ADO observed in this study and the effects of CH observed in the previous study suggests that
ADO does play an important role in the modulation of respiratory responses in the cane toad
during CH.
41
Chapter 4
The effects of a Midbrain Transection on Fictive Breathing
42
4.1 Introduction
The medulla oblongata is the site of endogenous respiratory rhythm generation in all vertebrates
including amphibians and mammals (McLean et al. 1995a; Reid et al. 2000a; Torgerson et al.,
1998; Wilson et al., 2002). However, breathing patterns can differ substantially amongst
different groups of vertebrates and can differ within one class of vertebrates under different
conditions. In mammals, birds and water-breathing fish, breathing is almost always rhythmic and
continuous although there are conditions such as hibernation and torpor in which breathing may
become discontinuous (Milsom, 1991). In contrast, breathing in adult amphibians is frequently
discontinuous and intermittent although it may become continuous under certain conditions
(Milsom, 1991). While discontinuous breathing in mammals, under non-pathological conditions,
is due to differential and mismatched input to the brain from oxygen and carbon dioxide
chemoreceptors, the intermittent pattern of breathing in adult amphibians can be independent of
arterial blood-gas levels. Under these conditions, discontinuous breathing is an intrinsic property
of the central respiratory control system (Kinkead, 1997).
Central respiratory groups involved in the control of mammalian breathing have been well
studied. On the other hand, fewer studies have identified specific sites in the brain that are, or
may be, involved with the control of breathing in amphibians. Some of these sites include the
nucleus isthmi (NI) (Kinkead et al., 1997; Gargaglioni and Branco, 2004; Milsom et al., 2004),
the locus coeruleus (LC) (Noronhade-Souza et al., 2006) and the Kölliker Fuse (Adli et al.,
1999). Two of these specific sites (i.e. the locus coeruleus and the nucleus isthmi) are found in
the midbrain, which has been implicated by several studies as an important region of the brain
that regulates the clustering of breaths into episodes (Oka, 1958a,b; Reid et al., 2000a;
McAneney and Reid, 2007; Milsom et al., 1999).
In anuran amphibians the locus coeruleus is described as a group of cells that innervate the spinal
cord, cerebellum and telencephalon (Parent, 1975; González and Smeets, 1991; 1993). These
cells contain noradrenaline (González and Smeets, 1991, 1993) and can be found in the isthmus
region located at the rostral end of the hindbrain (González and Smeets, 1991). Due to their
position, noradrenergic content and projections to both the telencephalon and spinal cord, the
locus coeruleus of the anuran amphibian is believed to be homologous to the locus coeruleus
found in mammals (Marin et al., 1996). The homology is supported by the study by Noronhade-
43
Souza et al. (2006) which demonstrated that just like the locus coeruleus in mammals, the locus
coerulues neurons in amphibians acts as a chemosensitive site in the CNS that modulates central
chemoreceptor information. As such, the locus coeruleus of the midbrain in the amphibian is
believed to contain CO2/H+-sensitive neurons involved primarily with central chemosensitivity
rather than the generation of the respiratory rhythm (Gargaglioni and Branco, 2009; Gargaglioni
et al., 2010).
Studies by Oka (1958a, b) demonstrated that transecting the brain behind the optic lobes (i.e.,
behind the midbrain) just in front of the cerebellum eliminated the episodic breathing pattern in
the Japanese bullfrog. Further studies on American bullfrogs have isolated the production of
episodic breathing patterns to the areas located in the caudal half of the midbrain (Milsom et al.,
1999; Reid et al., 2000a; Gargaglioni et al., 2007). The nucleus isthmi (NI) is a mesencephalic
structure found within the caudal half of the midbrain (between the roof of the midbrain and the
cerebellum). Although it was hypothesised that the NI is responsible for the generation of
episodic breathing in anuran amphibians (Kinkead et al., 1997), subsequent studies that involved
the manipulation of the NI, either by lesion and drug microinjections, disproved the notion that
the NI is directly responsible for turning episodic breathing on and off (Kinkead et al. 1997;
Gargaglioni and Branco 2000, 2001, 2003; Gargaglioni et al., 2002). Thus the specific area
responsible for episodic breath generation in amphibians remains unknown.
4.2 Hypothesis & Objectives
Previous studies (Reid et al., 2000; McAneney and Reid. 2007) have demonstrated that
transection of the midbrain of isolated brainstem-spinal cord preparations from bullfrogs and
cane toads reduces the episodic nature of fictive breathing and causes an increase in fictive
breathing frequency. The implication of these results is that central descending inputs from the
midbrain to the medulla function to cluster breaths into episodes and to reduce breathing
frequency. Furthermore, it has been suggested that the clustering of breaths into episodes
separated by periods of apnea involves alternating excitatory and inhibitory inputs from the
midbrain.
The experiments in this current chapter (chapter 4) and the next chapter (chapter 5) address the
hypothesis that the adenosine-mediated reduction in fictive breathing reported in chapter 3 is a
result of adenosine-mediated inputs from the midbrain into the medulla. In other words, the
44
adenosine-mediated reduction in breathing occurs due to physiological processes originating in
the midbrain. In the current chapter (chapter 4), the results of midbrain transection alone are
presented while in the next chapter (chapter 5) the effects of midbrain transection coupled with
pharmacological manipulation of adenosine A1R receptors are reported. In the current chapter, I
predict that a midbrain transection will reduce the episodic nature of fictive breathing (under
hypercapnic conditions where episodic breathing is expressed) and cause an increase in fictive
breathing frequency.
45
4.3 Materials and Methods
The general methods (surgery; brainstem-spinal cord preparation) are described in chapter 2.
Figure 4.1 illustrates the experimental protocol used in this chapter. Briefly, after a stabilisation
period, the preparation was exposed to various aCSF pH levels. Following this, the brainstem
was transected slightly caudal to the optic chiasma and the preparation was allowed to stabilise
for a 40 minute period. The transected preparation was then exposed to aCSF levels of varying
pH.
Figure 4.1: Overview of the stages and general procedures in the midbrain transection experiment. The experiment
is continuous and consists of two phases; (1) the pre-treatment phase in which the brainstem preparations are intact
aCSF and (2) the post-treatment phase following the midbrain transection. The pre-treatment phase of the
experiment is divided into two parts; (1) the stabilisation period during which the brainstem preparation is
superfused with aCSF at constant pH 7.8 and (2) the aCSF pH altering period during which the preparation is
exposed to random alterations of aCSF pH (15 minute duration for each pH change). The post-treatment phase of the
experiment is also divided into two parts; (1) the transection stabilisation period following the midbrain transection
during which the preparation is superfused with aCSF at constant pH of 7.8 for 40 minutes and (2) the transection
and aCSF pH altering period in which the transected brainstem preparation is exposed to aCSF of different pH levels
(15 minute duration for each pH change).
46
4.4 Data Analysis
The effects of altering the aCSF pH in the pre-transection and post-transection periods were
evaluated using a one-way repeated measures analysis of variance followed by a Holm-Sidak
multiple comparison test (MCT) The values during the post-transection stabilisation period (at
the constant aCSF pH of 7.8) were compared to the values during the pre-transection
stabilisation period using a paired t-test. A two-way ANOVA followed by a Holm-Sidak MCT
was used to evaluate the combined effect of aCSF pH and pre-transection / post-transection. The
limit of significance was 5% (p < 0.05), and all data are expressed as the mean ± one standard
error of the mean (S.E.M).
47
4.5 Results
4.5.1 Fictive Breathing Frequency
During the post-treatment stabilisation period (Fig. 4.2A), transecting the midbrain caused an
augmentation of fictive breathing frequency compared to that in preparations that had not had the
brainstem transected (p = 0.046).
Following the stabilisation period, the aCSF pH was changed from 7.8 to either 7.4, 7.6, 7.8 or
8.0. In the control group (Fig. 4.2B), fictive breathing frequency was increased at aCSF pH 7.4
(p = 0.001) and reduced at aCSF pH 8.0 (p = 0.004) compared to the values recorded at pH 7.8
(during the stabilisation period). In groups with the midbrain transected, fictive breathing
frequency decreased at an aCSF pH of 8.0 compared to the value at 7.8 during the stabilisation
period (Fig. 4.2B; p = 0.005). More importantly however, during the aCSF-altering phase of the
experiment (Fig. 4.2B), transecting the midbrain augmented fictive breathing frequency at aCSF
pH levels of 7.4 (p = 0.006) and 7.6 (p = 0.011); there was no effect at pH 7.8 and 8.0.
48
Figure 4.2: The effect of transecting the midbrain on fictive breathing frequency. (A) Fictive breathing frequency
during the stabilisation period (at a constant aCSF pH of 7.8). (B) Fictive breathing frequency at various aCSF pH
levels during the pH-altering phase of the experiment following the stabilisation period. A number sign (#) indicates
a significant difference between the treatment and control values. The data are reported as the mean ± S.E.M. An
asterisk (*) indicates a significant difference within a group at any given pH level compared to that at pH 7.8.
49
4.5.2 Fictive Episodes per Minute
During the stabilisation period at an aCSF pH of 7.8, the number of fictive breathing episodes
per minute was increased, compared to the control group (no brainstem transection), in response
to the midbrain transection (Fig. 4.3A; p = 0.043).
In the control group (Fig. 4.3B) lowering the aCSF pH to 7.4 (<0.001) and 7.6 (p = 0.036)
caused an increase in the number of fictive episodes per minute while raising pH to 8.0 caused a
reduction in episodes per minute (p = 0.001). In groups with the midbrain transected, there were
no significant differences in the number of fictive episodes per minute at any aCSF pH level
during the aCSF-altering phase when compared to a pH level of 7.8 during the stabilisation
period. More importantly however, during the aCSF-altering phase of the experiment (Fig.
4.3B), transecting the midbrain caused an augmentation in the number of fictive episodes per
minute when compared to controls at pH levels of 7.4 (p = 0.030) and 7.6 (p = 0.028) but not 7.8
and 8.0.
50
Figure 4.3: The effect of transecting the midbrain on the number of fictive breathing episodes per minute. (A) The
number of fictive episodes per minute during the stabilisation period (at a constant aCSF pH of 7.8). (B) The number
of fictive episodes per minute at various aCSF pH levels during the pH-altering phase of the experiment following
the stabilisation period. A number sign (#) indicates a significant difference between the treatment and control
values. An asterisk (*) indicates a significant difference within a group at any given pH level compared to that at pH
7.8 (during the stabilisation period). The data are reported as the mean ± S.E.M.
51
4.5.3 Fictive Breaths per Episode
There was no effect of transecting the brainstem on the number of fictive breaths per episode
either during the stabilisation period (Fig. 4.4A; p = 0.754) or during the pH-altering period (Fig.
4.4B; p = 0.689). There was no effect of altering aCSF pH on the number of fictive breaths per
episode in either the control group (Fig. 4.4B; p = 0.383) or the transected group (Fig. 4.4B; p =
p = 0.510).
4.5.4 Integrated area of fictive breaths
There was no effect of transecting the brainstem on fictive breath area either during the
stabilisation period (Fig. 4.5A; p = 0.953) or during the pH-altering period (Fig. 4.4B; p =
0.882). There was no effect of altering aCSF pH on fictive breath area in either the control group
(Fig. 4.5B; p = 0.491) or the transected group (Fig. 4.5B; p = 0.821).
4.5.5 Fictive Breath Duration
There was no effect of transecting the brainstem on fictive breath duration either during the
stabilisation period (Fig. 4.6A; p = 0.118) or during the pH-altering period (Fig. 4.6B; p =
0.451). There was no effect of altering aCSF pH on the number of fictive breath duration in the
transected group (Fig. 4.6B; p =0.868). However, the values of fictive breath duration during the
aCSF pH-altering phase were significantly larger in control groups (Fig 4.6B) at pH 7.4 (p =
0.004), 7.8 (p = 0.012) and 8.0 (p = 0.012) in comparison to the value observed during the
stabilisation period.
52
Figure 4.4: The effects of transecting the midbrain on the number of fictive breaths per episode. (A) The number of
fictive breaths per episode during the stabilisation period (at a constant aCSF pH of 7.8). (B) The number of fictive
breaths per episode at various aCSF pH levels during the pH-altering phase of the experiment following the
stabilisation period. The data are reported as the mean ± S.E.M.
53
Figure 4.5: The effects of transecting the midbrain on fictive breath area. (A) Fictive breath area during the
stabilisation period (at a constant aCSF pH of 7.8). (B) Fictive breath area at various aCSF pH levels during the pH-
altering phase of the experiment following the stabilisation period. The data are reported as the mean ± S.E.M.
54
Figure 4.6: The effects of transecting the midbrain on fictive breath duration. (A) Fictive breath duration during the
stabilisation period (at a constant aCSF pH of 7.8). (B) Fictive breath duration at various aCSF pH levels during the
pH-altering phase of the experiment following the stabilisation period. The data are reported as the mean ± S.E.M.
An asterisk (*) indicates a significant difference within a group at any given pH level compared to that at pH 7.8
(during the stabilisation period). The data are reported as the mean ± S.E.M.
55
4.5.6 Total Fictive Ventilation
During the stabilisation period, at a constant aCSF pH of 7.8, following brainstem transection,
total fictive ventilation was not different in the transected preparations than in the control non-
transected preparations (Fig. 4.7A; p = 0.165). In the subsequent pH altering phase of the
experiment (Fig. 4.7B), total fictive ventilation was greater in the transected preparations,
compared to the controls, at aCSF pH levels of 7.4 (p = 0.041) and 7.6 (p = 0.022). Total fictive
ventilation increased as aCSF pH was lowered in the control group at pH levels 7.4 (p = 0.002)
and decreased as aCSF pH was raised to pH level 8.0 (p =0.001) compared to the value at 7.8. In
groups with the midbrain transected, TFV decreased at an aCSF pH of 8.0 compared to the value
at 7.8 during the stabilisation period (Fig. 4.7B; p<0.001).
56
Figure 4.7: The effects of transecting the midbrain on total fictive ventilation. (A) Total fictive ventilation during
the stabilisation period (at a constant aCSF pH of 7.8). (B) Total fictive ventilation at various aCSF pH levels during
the pH-altering phase of the experiment following the stabilisation period. A number sign (#) indicates a significant
difference between the treatment and control values. An asterisk (*) indicates a significant difference within a group
at any given pH level compared to that at pH 7.8. The data are reported as the mean ± S.E.M.
57
4.6 Discussion
The current study was able to demonstrate that transecting at the level of the midbrain resulted in
an augmentation of total fictive ventilation (TFV) which was mediated by an augmentation of
fictive breathing frequency which, in turn, was caused by an elevation in the number of episodes
per minute. The augmentation in fictive breathing frequency and the number of episodes per
minute was manifest during both the stabilisation period at pH 7.8 as well as during the aCSF-
altering period at pH 7.4 and 7.6. The augmentation of TFV was only manifest during the pH-
altering period at the hypercapnic pH levels of 7.4 and 7.6 but not at the normocapnic or
hypocapnic pH levels of 7.8 and 8.0, respectively, while not identical, these are more-or-less
consistent with the study by McAneney and Reid (2007), which suggest that descending inputs
from the midbrain do attenuate respiratory motor output and that this attenuation is more likely
to occur during elevated respiratory drive.
Of relevance to this finding, although contradictory to the current results, is the study by
Gargaglioni et al. (2002) which demonstrated that chemical lesions to the NI in the toad
enhanced the ventilatory response to hypercapnia (3% inspired CO2). However, a chemical
lesion targeting a single brain centre is not analogous to the entire mid-brain transection
performed in this current study. In a subsequent study, Gargaglioni and Branco (2003) were able
to demonstrate that L-glutamate and NO within the NI have no role in respiration under resting
conditions and that L-glutamate and NO exert an inhibitory modulation on the hypoxic and
hypercapnic drives to breathe. This is consistent with a past study by McAneney et al. (2006),
which demonstrated that NMDA receptor-mediated mechanisms normally inhibit the increase in
breathing frequency associated with acute hypoxia. Collectively, these findings suggest that the
transection in the current study may have compromised L-glutamate and NO mediated
mechanisms which in turn lead to increases in breathing at hypercapnic pH levels in transected
preparations.
Both the current study and the study by McAneney and Reid (2007) did not transect the NI,
rather the transection was made in an area more rostral to the NI. In addition, the transection in
the current study as mentioned in chapter 2 (Fig. 2.3) was made slightly more rostral to the
transection site described by McAneney and Reid (2007). Regardless, both studies observed an
increase in fictive breathing frequency in response to the transection. This could be explained by
58
the fact that respiratory neurons of the brainstem can receive modulatory synaptic input from
non-respiratory regions such as the motor cortex, pontine and medullary reticular formations,
cerebellum, hypothalamus, other limbic and cardiovascular regions of the brainstem as well as
from extrapyramidal motor areas (Lalley, 2008), which function to adapt breathing rhythm and
pattern for effective cardio-respiratory interactions and activities (Feldman and McCrimmon,
2003). Hence, the findings in the current study suggest that the inhibitory inputs to ventilation
from the midbrain to the medulla are not limited to the caudal half of the midbrain.
One discrepancy between the study by McAneney and Reid (2007) and the current study was
that the midbrain transection did not switch the breathing pattern permanently, i.e. from
characteristic episodic breathing to continuous breathing. In the current study, continuous
breathing did occur but only immediately after transecting the midbrain, reverting back to
episodic breathing usually within the first forty minutes of transection (refer to the appendix, Fig.
A.8).Since the transection in the current study was done via an incision with a pair of Westcott
scissors, the cells along the incision were most likely damaged. Within the cytosol that is
released from damaged cells are various chemical triggers, such as ATP and glutamate, which
can cause action potential firing (Cook and McClesky, 2002). Since breath pattern in the
amphibian can become continuous during extremely high levels of respiratory drive (Milsom,
1991; Gargaglioni and Milsom, 2007), it is possible that the brief change to a continuous breath
pattern may have been caused by the excitatory triggers within the cytosol released from
damaged cells (both of which are caused by high levels of excitation).
In current literature, the only known fact with regards to the site of episodic breath generation is
that it is found within the caudal half of the midbrain (Milsom et al. 1999; Reid et al. 2000;
Gargaglioni et al. 2007; McAneney and Reid, 2007). The current study supports these findings,
as the episodic breath pattern was still observable in the midbrain transected preparations that
was transected approximately through the middle of the midbrain rather than through the caudal
half as described in the study by McAneney and Reid (2007). The NI, which is a mesencephalic
structure that is found between the roof of the midbrain and the cerebellum, was proposed to be
that specific site of episodic breath generation (Kinkead et al., 1997). However a study by
Gargaglioni and Branco (2000), demonstrated that an electrolytic lesion of the NI failed to
eliminate the episodic breath pattern. Hence, the specific site of episodic breath generation
within the caudal half of the amphibian midbrain still remains unknown.
59
Regardless, from the point-of-view of this thesis, the important result is that the midbrain
transection caused an increase in fictive breathing at hypercapnic aCSF pH levels (7.4 and 7.6).
This indicates that descending inputs from the midbrain to the medulla normally inhibit
breathing under these conditions of elevated respiratory drive. As such, the next step is to
determine if these inhibitory inputs are adenosinergic (adenosine-mediated) in nature. This is the
focus of the next chapter in this thesis.
60
Chapter 5
Effects of Adenosine, CCPA and DPCPX on Fictive Breathing Following a Midbrain Transection
61
5.1 Introduction
It has been suggested that respiratory-related midbrain input to the medulla in anuran amphibians
is generally inhibitory (Gargaglioni and Branco, 2003; McAneney and Reid, 2007). However,
the nature of those midbrain factor(s) that exert inhibitory influences on breathing remains
unknown. Gargaglioni and Branco (2003) suggested that the putative mediators within the
nucleus isthmi (NI), L-glutamate and NO, exert inhibitory modulation on breathing during
hypoxia and hypercapnia.
During chronic hypoxia (CH), the inhibition of breathing arising from these midbrain descending
inputs is enhanced (McAneney and Reid, 2007). However, other studies have shown that NMDA
receptor function becomes reduced but not eliminated in hypoxia-tolerant animals during CH
(Bickler, 2004; Buck, 2004; Downey et al., 2007;McAneney et al., 2006; Pagonopoulou et al.,
2006) and that Ca2+
influx via activation of NMDA receptors is a key trigger for NO production
(Garthwaite et al., 1988; 1989). Collectively, the findings suggest that L-glutamate and NO,
which may play a role in the inhibitory nature of the midbrain during elevated respiratory drive,
may not have a role in the inhibitory nature of the midbrain during CH. Given this, I speculated
that a more universal factor, adenosine, that is both present and functional during elevated
respiratory drive and CH, is responsible for the inhibitory modulation of breathing exerted by the
descending inputs from the midbrain.
Since the descending inputs from the midbrain exert inhibitory influences during conditions of
elevated respiratory drive, which becomes further enhanced during CH, it is reasonable to
assume that the possible factor(s) behind the inhibitory nature of the midbrain must (1) inhibit
respiratory activity, (2) be present during a variety of levels of respiratory drive and (3) be
modified in such a manner that its inhibitory effects are enhanced during CH. One such factor is
the metabolite adenosine (ADO). Chapter 3 of this thesis determined that adenosine and an
adenosine A1R agonist (CCPA) reduced fictive breathing whilst an adenosine A1R antagonist
(DPCPX) augmented fictive breathing. Chapter 4 of this thesis indicated that transection through
the midbrain caused an increase in fictive breathing at hypercapnic aCSF pH levels indicating
that descending inputs from midbrain to the medulla normally inhibit breathing under conditions
of elevated respiratory drive. In this current chapter, I hypothesize that the midbrain input to the
medulla is inhibitory due to ADO-mediated mechanisms and subsequent A1R activation within
62
the midbrain. If this is the case, then I predict that transecting the midbrain would reduce the
inhibition to breathing caused by ADO treatment, at least in comparison to the effects of ADO
on intact brainstem-cord preparations. This hypothesis was tested by comparing the effects of
ADO, CCPA and DPCPX during both a stabilisation period and a pH-altering period on fictive
breathing recorded from brainstem-spinal cord preparations with and without the midbrain
transected at the same level as described in chapter 4.
63
5.2 Materials and Methods
The brainstem-spinal cord preparations were obtained as described in chapter 2. Figure 5.1
illustrates the protocol used in these experiments.
Figure 5.1: Overview of the stages and general procedures used within the experiment on midbrain transected
preparations. The experiment was one continual process made up of two phases; (1) the pre-treatment phase in
which brainstem preparations were kept intact and exposed only to aCSF of varying pH levels. (2) the post-
treatment phase in which the brainstem preparation with the midbrain transected was exposed to a dose of ADO,
CCPA or DPCPX along with varying aCSF pH levels. The pre-treatment phase of the experiment was split into two
parts; (1) the stabilisation period in which the brainstem preparation was exposed to aCSF at a constant pH 7.8 and
(2) the pH treatment period in which the brainstem preparation was exposed to alterations in aCSF pH (15 minute
duration for each pH change). The post-treatment phase of the experiment was also split into two parts; (1) the
transection & dose stabilisation period during which the midbrain of the brainstem preparation was transected and
exposed to a new aCSF reservoir containing a dose of ADO or the A1R agonist, CCPA or the A1R antagonist,
DPCPX (kept at constant pH 7.8) for 40 minutes and (2) the transection (with ADO or CCPA or DPCPX ) and pH
treatment period during which the ADO or CCPA or DPCPX was still present and the transected brainstem
preparation was exposed to alterations in aCSF pH (15 minute duration for each pH change).
64
5.3 Data Presentation and Analysis
In figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12, panels A and B are the same figures (data) as
presented in chapter 3. They illustrate the effects of adenosine, CCPA and DPCPX on
preparations with the midbrain intact (no transection).
In figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12, panels C and D represent the effects of the
pharmacological agents (adenosine, CCPA and DPCPX) on preparations in which the midbrain
was transected (i.e., previously un-reported data). Panel C shows the data from the stabilisation
period and panel D shows the effects of changing the aCSF pH following the stabilisation period
(i.e., during the pH-treatment/varying period). Note, in these transection experiments, the 1µM
adenosine dose was not administered as the effects of both 1 and 10 µM adenosine with the
midbrain intact were identical. This was done to shorten the experimental protocol and reduce
the number of animals used. In panel C of these figures, “midbrain” refers to the stabilisation
period following midbrain transection with no pharmacological agent treatment. In panel D,
“midbrain transection” refers to preparations that had the midbrain transected but were not
treated with any pharmacological agents during the pH-altering period. In other words, it is
analogous to the “control” data in panel A.
A one-way repeated measures analysis of variance followed by a Holm-Sidak multiple
comparison test was used to compare the effects of controls (i.e., normal aCSF), adenosine,
CCPA and DPCPX during the 40 minute stabilisation period either with the midbrain intact or
the midbrain transected [i.e., the effects illustrated in panels A (midbrain intact) and C (midbrain
transected) of figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12].
A two-way ANOVA followed by a Holm-Sidak test was used to compare the effects of treatment
with the pharmacological agents and aCSF pH changes during the “pH-altering phase” of the
experiment [i.e., the effects illustrated in panels B (midbrain intact) and D (midbrain transected)
of figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12].
Figures 5.3, 5.5, 5.7, 5.9, 5.11 and 5.13 re-plot the data in panels B (midbrain intact) and D
(midbrain transected) of figures 5.2, 5.4, 5.6, 5.8, 5.10 and 5.12 in order to more readily observe
the effects of the different pharmacological agents with the midbrain intact and the midbrain
transected. As such, these figures show the effects of midbrain transection on fictive breathing
65
under control conditions (panel A) and in response to treatment with 10 µM ADO (panel B),
CCPA (panel C) or DPCPX (panel D).
A two-way ANOVA followed by a Holm-Sidak test was used to compare the effects of midbrain
transection and aCSF pH changes under control conditions and in response to treatment with
ADO, CCPA or DPCPX (i.e., the data in figures 5.3, 5.5, 5.7, 5.9, 5.11 and 5.13).
The limit of significance was 5% (p < 0.05), and all data are expressed as the mean ± one
standard error of the mean (S.E.M).
66
5.4 Results
5.4.1 Fictive Breathing Frequency
Note, panels A (stabilisation period) and B (aCSF pH-altering period) are the data collected from
preparations with the midbrain intact. They are the same data as reported in chapter 3. As such,
there is no written description of these figures in the subsequent text which focuses on those
preparations in which the midbrain was transected (i.e., the data in panels C and D).
During the dose stabilisation period of the experiment in which the midbrain was transected (Fig.
5.2C), perfusion of ADO (p = 0.140), or CCPA (p = 0.130) or DPCPX (p = 0.980) had no
significant effect on fictive breathing frequency.
During the pH-altering phase of the experiment on preparations with the midbrain transected
(Fig. 5.2D), superfusion with 10 µM ADO caused a decrease in fictive breathing frequency,
compared to the controls (no pharmacological agents) at aCSF pH levels of 7.4 (p < 0.001), 7.6
(p = 0.003) and 7.8 (p = 0.038). Superfusion with CCPA caused a reduction in fictive breathing
frequency at aCSF pH levels of 7.4 (p= 0.014) and 7.6 (p = 0.013). Superfusion with DPCPX
had no effect on fictive breathing frequency, compared to the controls, at any aCSF pH level (pH
7.4, p = 0.644; pH 7.6, p = 0.951; pH 7.8, p = 0.430; pH 8.0, p = 0.214).
Altering the aCSF pH had minor effects on fictive breathing in the various groups (see the
asterisks on Fig. 5.2D).
67
Figure 5.2: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R
antagonist (DPCPX; 10 µM) on fictive breathing frequency measured from brainstem-spinal cord preparations with
the midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures
as reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with
the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-
altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the
midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value
recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and
“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An
asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the
pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the
stabilisation period .
68
Figure 5.3 illustrates the effects of midbrain transection on the responses to ADO, CCPA,
DPCPX, as well as under control conditions, at various aCSF pH levels. Under control
conditions (Fig. 5.3A), fictive breathing frequency was elevated following a midbrain transection
at aCSF pH levels of 7.4 (p = 0.006) and 7.6 (p = 0.011). Fig. 5.3B illustrates that midbrain
transection increased fictive breathing frequency at aCSF pH levels of 7.4 (p < 0 .001) and 7.6 (p
= 0.0222) in response to ADO. Midbrain transection increased fictive breathing frequency at pH
levels 7.4 (p < 0.001), 7.6(p = 0.026) and 7.8 (p = 0.008) in response to CCPA (Fig. 5.3C).
Midbrain transection had no effect on the response to DPCPX treatment (Fig. 5.3D; p = 0.970).
69
Figure 5.3: Fictive breathing frequency recorded from brainstem-spinal cord preparations with the midbrain intact
(closed symbols) and the midbrain transected (open symbols) under control conditions (panel A) and following
treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a number
sign (#) indicates a significant difference between preparations with the midbrain intact and the preparations with
the midbrain transected.
70
5.4.2 Fictive Episodes per Minute
During the dose stabilisation period of the experiment, perfusion with ADO caused a significant
reduction in the number of fictive episodes per minute in midbrain transected preparations (Fig.
5.4C; p = 0.015). Unlike ADO, treatment with CCPA (p = 0.107) or DPCPX (p = 0.948) on
midbrain transected preparations did not have a significant effect on the number of fictive
episodes per minute during the dose stabilisation period (Fig. 5.4C).
During the pH-altering phase of the experiment (Fig. 5.4D), superfusion with ADO caused a
reduction in the number of fictive episodes per minute at all aCSF pH levels (pH, 7.4, p < 0.001;
pH 7.6, p < 0.001; pH 7.8, p = 0.041; pH 8.0, p = 0.022). Treatment with CCPA caused
reductions in the number of episodes per minute at pH levels of 7.4 (p = 0.003), 7.6 (p = 0.003)
and 8.0 (p = 0.047). In DPCPX-treated midbrain-transected brainstem preparations, no
significant differences in the number of fictive episodes per minute were observed at any of the
pH levels (p = 0.339). With the exception of pH 8.0 in the CCPA-treated group, alterations in
aCSF pH had no effect on the number of fictive episodes per minute.
Figure 5.5 illustrates that midbrain transection caused an increase in the number of fictive
episodes per minute under control conditions (Fig. 5.5A) at aCSF pH levels of 7.4 (p = 0.030)
and 7.6 (p = 0.028). The number of episodes per minute was also elevated in response to CCPA
treatment (Fig 5.5C) at aCSF pH levels of 7.4 (p = 0.006), 7.6 (p = 0.030) and 7.8 (p = 0.013).
Midbrain transection had no effect on the number of fictive breathing episodes during treatment
with ADO (Fig. 5.5B) or DPCPX (Fig. 5.5D).
71
Figure 5.4: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R
antagonist (DPCPX; 10 µM) on fictive episodes per minute measured from brainstem-spinal cord preparations with
the midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures
as reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with
the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-
altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the
midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value
recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and
“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An
asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the
pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the
stabilisation period .
72
Figure 5.5: The number of fictive breathing episodes per minute recorded from brainstem-spinal cord preparations
with the midbrain intact (closed symbols) and the midbrain transected (open symbols) under control conditions
(panel A) and following treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D).
In all panels a number sign (#) indicates a significant difference between preparations with the midbrain intact and
the preparations with the midbrain transected.
73
5.4.3 Fictive Breaths per Episode
During the dose stabilisation period of the experiment (Fig. 5.6C), perfusion of ADO (p =
0.428), CCPA (p = 0.160) or DPCPX (p = 0.953) did not affect the number of fictive breaths per
episode in brainstem preparations with the midbrain transected.
During the pH-altering phase of the experiment on midbrain-transected preparations (Fig. 5.6D),
treatment with ADO caused an increase in the number of fictive breaths per episode compared to
the controls at aCSF pH levels of 7.4 (p = 0.009) and 7.6 (p = 0.034). CCPA caused an increase
in the breaths per episode at pH 8.0 (p = 0.005).
Altering aCSF pH had no effect on the number of fictive breaths per episode in any of the groups
(Fig. 5.6D).
Figure 5.7 illustrates that midbrain transection had no effect on the number of breaths per
episode under control conditions (p = 0.689) or in response to treatment with ADO (p = 0.577),
CCPA (p = 0.207) or DPCPX (p = 0.184).
74
Figure 5.6: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R
antagonist (DPCPX; 10 µM) on fictive breaths per episode measured from brainstem-spinal cord preparations with
the midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures
as reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with
the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-
altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the
midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value
recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and
“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An
asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the
pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the
stabilisation period .
75
Figure 5.7: The number of fictive breaths per episode recorded from brainstem-spinal cord preparations with the
midbrain intact (closed symbols) and the midbrain transected (open symbols) under control conditions (panel A) and
following treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a
number sign (#) indicates a significant difference between preparations with the midbrain intact and the preparations
with the midbrain transected.
76
5.4.4 Integrated Area of Fictive Breaths
During the dose stabilisation period of the experiment (Fig. 5.8C), fictive breath area in the
midbrain-transected brainstem preparations was not affected by treatment with ADO (p = 0.669),
CCPA (p = 0.094) or DPCPX (p = 0.748). Similarly, during the pH-altering phase of the
experiment there was no effect of ADO (p = 0.938), CCPA (p = 0.304) or DPCPX (p = 0.186).
Fictive breath area was not affected by the alteration of aCSF pH levels in the control, adenosine
and DPCPX treated groups (Fig. 5.8D). However, the group treated with CCPA had a
significantly lower fictive breath area during the aCSF pH-altering phase at all pH levels than it
was during the stabilisation period (Fig. 5.8D; pH 7.4, p = 0.003; pH 7.6, p = 0.011; pH 7.8, p =
0.007; pH 8.0, p = 0.003; compare all values in Fig. 5.8D with the value in Fig. 5.8C).
Transection of the midbrain did not affect fictive breath area under control conditions (Fig. 5.9A;
p = 0.953) nor in response to CCPA (Fig. 5.9C; p = 0.302) or DPCPX (Fig. 5.9D; p = 0.142). In
response to ADO (Fig. 5.9B), midbrain transection caused an increase at pH 7.8 (p = 0.003) but
not at the other aCSF pH levels (pH 7.4, p = 0.061; pH 7.6, p = 0.075; pH 8.0, p = 0.305).
77
Figure 5.8: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R
antagonist (DPCPX; 10 µM) on fictive breath area measured from brainstem-spinal cord preparations with the
midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures as
reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with
the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-
altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the
midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value
recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and
“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An
asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the
pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the
stabilisation period .
78
Figure 5.9: Fictive breath area recorded from brainstem-spinal cord preparations with the midbrain intact (closed
symbols) and the midbrain transected (open symbols) under control conditions (panel A) and following treatment
with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a number sign (#)
indicates a significant difference between preparations with the midbrain intact and the preparations with the
midbrain transected.
79
5.4.5 Fictive Breath Duration
Fictive breath duration observed in midbrain-transected preparations was not affected during the
stabilisation period (Fig 5.10C) during treatment with ADO (p = 0.826) or CCPA (p = 0.075) or
DPCPX (p = 0.050).
During the pH-altering phase of the experiment (Fig. 5.10D), treatment with ADO caused an
increase in fictive breath duration, compared to the control values at aCSF pH levels of 7.4 (p <
0.001) and 7.8 (p = 0.006). In CCPA treatments, fictive breath duration in midbrain transected
preparations was significantly augmented at all aCSF pH levels (Fig. 5.10D; pH 7.4, p = 0.004;
pH 7.6, p = 0.006; pH 7.8, p < 0.001; pH 8.0, p = 0.001). On the other hand, DPCPX treatment
did not significantly affect fictive breath duration at any aCSF pH level (Fig. 5.10D; pH 7.4, p =
0.092; pH 7.6, p = 0.066; pH 7.8, p = 0.216; pH 8.0, p = 0.211).
When compared to the values recorded at pH 7.8 in the stabilisation period (Fig. 5.10B), fictive
breath duration in response to ADO (Fig. 5.10D) was significantly larger during the aCSF pH-
altering phase at all pH levels (pH 7.4, p = 0.001; pH 7.6, p = 0.012; pH 7.8, p < 0.001; pH 8.0, p
= 0.007). In response to CCPA treatment, (Fig 5.10D), the value of fictive breath duration was
larger when compared to that observed in the stabilisation period at pH 7.8 (p = 0.044).
80
Figure 5.10: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R
antagonist (DPCPX; 10 µM) on fictive breath duration measured from brainstem-spinal cord preparations with the
midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures as
reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with
the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-
altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the
midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value
recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and
“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An
asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the
pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the
stabilisation period .
81
In the control group (Fig. 5.11A), there was no effect of transecting the midbrain on fictive
breath duration (p = 0.451). However, midbrain transection led to significant increases in fictive
breath duration in response to ADO (Fig 5.11B), CCPA (Fig 5.11C) and DPCPX (Fig. 5.11D)
treatment. Following ADO treatment (Fig 5.11B), fictive breath duration in midbrain transected
brainstem preparations was significantly greater than in intact preparations at aCSF pH levels of
7.4 (p = 0.019), 7.6 (p = 0.028) and 7.8 (p = 0.004). Similarly, superfusion with CCPA (Fig.
5.11C) significantly increased fictive breath duration in midbrain transected brainstem
preparations compared to intact brainstem preparations at all aCSF pH levels (pH 7.4, p < 0.001;
pH 7.6, p < 0.001; pH 7.8, p < 0.001; pH 8.0, p < 0.001). In DPCPX-treated transected brainstem
preparations (Fig. 5.11D), fictive breath duration was significantly elevated compared to intact
brainstem preparations at pH 7.6 (p = 0.014) and 8.0 (p = 0.012).
82
Figure 5.11: Fictive breath duration recorded from brainstem-spinal cord preparations with the midbrain intact
(closed symbols) and the midbrain transected (open symbols) under control conditions (panel A) and following
treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a number
sign (#) indicates a significant difference between preparations with the midbrain intact and the preparations with
the midbrain transected.
83
5.4.6 Total Fictive Ventilation
Total fictive ventilation (TFV) was not affected in midbrain-transected brainstem preparations in
response to treatment with ADO (p = 0.348), CCPA (p = 0.395) or DPCPX (p = 0.727) during
the dose stabilisation period of the experiment (Fig. 5.12C).
During the pH-altering phase of the experiment (Fig. 5.12D), ADO treatment caused a
significant reduction in TFV at pH 7.4 (p = 0.022) and 7.6 (p = 0.033) while CCPA treatment
caused a significant reduction at pH 7.4 (p = 0.024). DPCPX had no effect (p = 0.879).
In the CCPA group, TFV during the pH-altering phase of the experiment (Fig. 5.12D) was less
than the value observed at pH 7.8 during the stabilisation phase at all aCSF pH levels (pH 7.4, p
= 0.004; pH 7.6, p = 0.003; pH 7.8, p = 0.001; pH 8.0, p < 0.001). There was also a significant
decrease in the control group at pH 8.0 (p = 0.017). There was no effect of DPCPX on the
response to altered pH levels.
Figure 5.13 illustrates the effects, on TFV, of transecting the midbrain under control conditions
and in response to the various pharmacological agents. Under control conditions (Fig. 5.13A),
transection led to increases in TFV at pH levels of 7.4 (p=0.041) and 7.6 (p = 0.022). In
midbrain-transected preparations that were treated with ADO (Fig. 5.13B) or CCPA (Fig.
5.13C), TFV was significantly greater in midbrain-transected brainstem preparations compared
to intact preparations at aCSF pH levels 7.4 (ADO, p = 0.008; CCPA, p = 0.023), 7.6 (ADO, p =
0.016; CCPA, p = 0.037) and 7.8 (ADO, p = 0.014; CCPA, p = 0.029). Conversely, transecting
the midbrain did not affect TFV in response to DPCPX at any pH level (Fig. 5.13D; p = 0.401).
84
Figure 5.12: The effects of adenosine (1 and 10 µM or10 µM only), an A1R agonist (CCPA, 10 µM) and an A1R
antagonist (DPCPX; 10 µM) on total fictive ventilation measured from brainstem-spinal cord preparations with the
midbrain intact (A and B) and those with the midbrain transected (C and D). Panels A and B are the same figures as
reported in chapter 3. Panels A and C represent data recorded during the 40 minute stabilisation period either with
the midbrain intact (panel A) or transected (panel C). Panels B and D represent the data recorded during the pH-
altering phase of the experiment that followed the stabilisation period with the midbrain intact (panel B) or the
midbrain transected (panel D). In all panels a number sign (#) indicates a significant difference between the value
recorded with no pharmacological agent in the aCSF (i.e., “control” in panels A and B; “midbrain” in panel C and
“midbrain transection” in panel D) and the values recorded with the various pharmacological manipulations. An
asterisk (*) indicates a significant difference between the value of a respective group at a particular pH during the
pH manipulation phase compared to the value of the same respective group recorded at pH 7.8 during the
stabilisation period .
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Figure 5.13: Total fictive ventilation recorded from brainstem-spinal cord preparations with the midbrain intact
(closed symbols) and the midbrain transected (open symbols) under control conditions (panel A) and following
treatment with 10 µM adenosine (panel B), 10 µM CCPA (panel C) or 10 µM DPCPX (D). In all panels a number
sign (#) indicates a significant difference between preparations with the midbrain intact and the preparations with
the midbrain transected.
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5.5 Discussion
The experiments in chapter 3 illustrated that treatment with ADO and the A1R agonist, CCPA
caused a decrease in total fictive ventilation (TFV) mediated by a decrease in fictive breathing
frequency. Treatment with the A1R antagonist DPCPX caused an increase in fictive breathing
frequency which led to a non-statistically significant increase in TFV due to a concomitant
decrease in fictive breath area; TFV being the product of frequency and area. The conclusion
from that chapter is that adenosine-mediated mechanisms cause a reduction in fictive breathing
in the isolated brainstem-spinal cord preparations. Logic suggests that similar mechanisms would
lead to a decrease in breathing in the intact animal.
The results of chapter 4 indicate that transection of the midbrain leads to an increase in fictive
breathing. The interpretation of these results is that input from the midbrain to the respiratory
centres in the medulla oblongata normally result in a reduction in breathing since their removal
by transection leads to increases in breathing. The current chapter addressed the hypothesis that
these descending influences are, at least in part, mediated by adenosine-mediated mechanisms
(i.e., neurotransmission or neuromodulation).
5.5.1 Possible Sites of Adenosine Action
Superfusion of adenosine or CCPA onto the brainstem-spinal cord preparation with the midbrain
intact could lead to decreases in fictive breathing via several pathways. First, ADO or CCPA
may stimulate adenosine receptors (all types in the case of ADO and A1R in the case of CCPA)
within respiratory centres in the medulla. These respiratory centres may or may not be innervated
by adenosinergic (purinergic) neurons originating from the midbrain although my hypothesis
suggests that they are. Second, ADO or CCPA may stimulate neurons in the midbrain which, in
turn, activate other neurons that are non-purinergic in nature that then descend and innervate
respiratory centres within the medulla. Treatment with the A1R antagonist DPCPX would be
expected to antagonise the effects of endogenous adenosine regardless of the anatomical location
of the receptors.
In current literature, the distribution of ADO receptors in the brain has been documented in a
variety of mammals (Goodman and Snyder, 1982; Fastbom et al., 1987; Dixon et al., 1996;
Naganawa et al., 2006) but not in anuran amphibians. The distribution of the A1Rs have been
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found throughout the mammalian brain in areas such as the cortex, amygdala, striatum, olfactory
bulb, nucleus accumbens, hippocampus, hypothalamus, thalamus and cerebellum.
In this current chapter I hypothesized that the midbrain input to the medulla is inhibitory due to
ADO-mediated mechanisms and subsequent A1R activation within the midbrain. If this is the
case then I predict that transecting the midbrain would reduce the inhibition to breathing caused
by ADO/CCPA treatment compared to the effects of ADO/CCPA on intact brainstem-cord
preparations.
5.5.2 The Effects on Adenosine, CCPA and DPCPX
Total fictive ventilation (TFV) is the global measure of breathing recorded from the in vitro
brainstem-spinal cord preparations. A comparison of the effects of ADO, CCPA and DPCPX
with the midbrain intact (Fig. 5.12A) and the midbrain transected (Fig. 5.12C) during the dose
stabilisation period at an aCSF pH of 7.8 indicates that the ADO- and CCPA-mediated decreases
in breathing that occur with the midbrain intact are abolished when the midbrain is transected.
The interpretation of this result is that the adenosine-mediated mechanisms that cause a reduction
in breathing originate from the midbrain albeit at a normocapnic pH of 7.8. This result is
corroborated during the pH-altering phase of the experiment (compare Figs. 5.12C and D) as the
ADO- and CCPA-mediated decreases in TFV with the midbrain intact were abolished when the
midbrain was transected; again at a normocapnic pH of 7.8. As such, the data leads to the
conclusion that adenosine-mediated mechanisms acting through the A1 purinergic receptor
somewhere in the rostral midbrain cause decreases in breathing under normocapnic conditions.
However, removal of the midbrain influences via transection did not prevent the ADO- or
CCPA-mediated decreases in TFV at hypercapnic pH levels. At pH 7.4 and 7.6, ADO treatment
still led to a decrease in TFV following midbrain transection whereas at pH 7.4 CCPA treatment
still led to a decrease in TFV following midbrain transection. These results suggest that the
inhibition of breathing by adenosine-mediated mechanisms under hypercapnic conditions occurs
somewhere other than in the midbrain; presumably within respiratory centres in the medulla.
With the exception of the data at pH 8.0 with the midbrain intact, blocking the actions of
endogenously produced adenosine by superfusing the brainstem-spinal cord preparations with
DPCPX had no effect on total fictive ventilation either with the midbrain intact (Fig. 5.12B) or
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following a midbrain transection (Fig. 5.12C). However, treatment with DPCPX did cause a
significant increase in fictive breathing frequency (Fig. 5.2A) and a significant decrease in fictive
breath area (Fig. 5.8A) during the stabilisation phase of the experiment (pH 7.8) with the
midbrain intact. The interpretation of these results is that under normocapnic conditions (pH 7.8),
adenosine-mediated mechanisms normally function to reduce breathing frequency and increase
breath amplitude (an index of breath volume; the equivalent of mammalian tidal volume). A
similar effect was observed at pH 7.8 (and indeed at all aCSF levels) during the pH-altering
phase of the experiment for breathing frequency (Fig. 5.2B) but not for breath area/amplitude
(Fig. 5.8B).
During the pH-altering phase with the midbrain intact (Fig. 5.2B), DPCPX caused an increase in
fictive breathing frequency under hypercapnic (7.4; 7.6), normocapnic (7.8) and hypocapnic
(8.0) pH levels indicating that endogenous adenosine-mediated mechanism working via A1
receptors function to reduce breathing frequency regardless of the pH/CO2-induced drive to
breathe. These effects of DPCPX were abolished following the midbrain transection (compare
the effects of DPCPX in Figs. 5.2B and D). However, the abolition of the DPCPX-induced
increase in breathing frequency following midbrain transection is due to the fact that the
midbrain transection itself elevated fictive breathing frequency in the control group but had no
effect in the DPCPX group (see Fig. 5.3A and D). The simplest explanation is that the midbrain
transection has removed the A1 receptors that were being influenced by DPCPX with the
midbrain intact. In other words, there are A1 receptors in the midbrain that normally cause a
reduction in breathing. Their effect can be removed either by treating with DPCPX with the
midbrain intact or by removing the rostral portion of the midbrain. With the rostral portion of the
midbrain gone, DPCPX had no further effect.
Alternatively, the augmentation to fictive breathing frequency following DPCPX treatment could
have been caused by the endogenous activation of stimulatory ADO receptors (A2A and A2B
receptors). Assuming the A1Rs are fully antagonized by DPCPX, the A2A receptors followed by
the A2B receptors have the next highest affinity for ADO. If this were the case, then one
possibility pertaining to the abolished response to DPCPX in midbrain-transected preparations is
that transecting the midbrain removed input from the striatum, olfactory tubercle, hypothalamus
and nucleus accumbens, which are areas that are highly concentrated with stimulatory A2A
receptors in the mammalian brain (Dixon et al., 1996; Mishina et al., 2007). Repeating the
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experimental procedure described in the current study but with A2A, A2B or A3 agonist/antagonist
treatment of midbrain-transected preparations could provide further insight as to whether
breathing is influenced by other types of adenosine receptors and whether the effects are altered
by removal of midbrain influences.
In the current study, DPCPX (A1R antagonist) and CCPA (A1R agonist) treatment demonstrated
that the transection of the midbrain does not eliminate adenosine-mediated modulation through
the A1R. Since the transected preparations remain responsive to CCPA, this suggests that A1R
are as diversely distributed in the brain of amphibians as they are in mammals. If A1Rs are
diversely distributed in the brain of cane toad, then any decrease in A1R efficiency by the
midbrain transection could be caused by the elimination of A1R regulation originating from the
midbrain and rostral brain sites, which would support the hypothesis that the descending inputs
from the level of the midbrain suppresses respiratory activity through purinergic regulation of
ADO primarily through the A1R. Indeed preliminary data (Peters and Reid, in preparation) from
RT-PCR experiments indicate that the A1R is, as expected, present in whole brain extracts from
the cane toad. Experiments are on-going to isolate the presence of these receptors to regions of
the midbrain and medulla. In addition, in vitro light microscopic autoradiographic methods
(Goodman and Snyder, 1982) could help verify the relative densities of ADO receptors in the
brain of cane toad.
The regulation of fictive breath duration in response to adenosine-mediated mechanisms appears
to be an intrinsic function of the midbrain. In intact preparations, 10 μM ADO caused a reduction
in duration at pH 7.8 during the stabilisation period but had no effect during the pH-altering
period while CCPA failed to alter breath duration. Antagonizing the A1R with DPCPX on intact
preparations caused an overall decrease in fictive breath duration during the pH-altering period.
The data therefore appear contradictory. The interpretation of the DPCPX data alone is that A1R-
mediated mechanisms normally cause an increase in breath duration (because it decreases when
the receptors are blocked with DPCPX). However, if A1R activation normally increases breath
duration then one might expect that the ADO and CCPA treatment would have caused increases
in breath duration compared to the controls. The fact that this did not occur suggests that the
duration of the fictive breaths under control conditions is already at a maximal level and that
further stimulation of A1R mediated mechanisms had no further effect (putting aside the effect
observed in Fig. 5.10A).
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After transecting the midbrain, fictive breath duration increased significantly in response to ADO
and CCPA (see Fig. 5,11B and C). In other words, ADO and CCPA had no effect on fictive
breath duration (aside from the single anomalous result in Fig. 5.10A) with the midbrain intact
but caused increases when the midbrain was transected. Collectively, the results suggest that the
A1R activity functions to augment breath duration but is confined to a certain degree that is
governed by the input from midbrain, the nature of which cannot be surmised from the results of
this study.
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Chapter 6
Summary, Conclusions and General Discussion
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6.1 Summary of the Major Results of the Thesis
The major results of chapter 3 are as follows: 1) With the midbrain intact, treatment with
adenosine (1 and 10 µM) and an adenosine A1 receptor (A1R) agonist (10 µM CCPA) caused a
reduction in total fictive ventilation (TFV: chapter 3, figure 3.7) 2) The effects of ADO and
CCPA on TFV were due to reductions in fictive breathing frequency (chapter 3, figure 3.2)
rather than a reduction in fictive breath area (chapter 3, figure 3.5). 3) The effects of ADO and
CCPA on fictive breathing frequency were due to reductions in the number of fictive episodes
per minute (chapter 3, figure 3.3) rather than the number of fictive breaths per episode (chapter
3, figure 3.4). 4) With the midbrain intact, treatment with an A1R antagonist (10 µM DPCPX)
had no effect on TFV (chapter 3, figure 3.7). However, DPCPX did cause an increase in fictive
breathing frequency (chapter 3, figure 3.2) mediated by an increase in the number of fictive
breathing episodes per minute (chapter 3, figure 3.3) and a decrease in fictive breath area
(chapter 3, figure 3.5). 5) With the midbrain intact, the effects of ADO, CCPA and DPCPX were
manifest predominately at normocapnic (7.8) and hypercapnic (7.4 and 7.4) aCSF pH levels.
The major results of chapter 4 are as follows: 1) Transection of the midbrain to remove the
rostral regions caused an increase in TFV at hypercapnic aCSF pH levels (7.4 and 7.6; chapter 4,
figure 4.7) that was mediated by an increase in fictive breathing frequency (chapter 4, figure 4.2)
which in turn was mediated by an increase in the number of fictive episodes per minute (chapter
4, figure 4.3). 2) Despite causing an increase in TFV, the midbrain transection did not abolish the
discontinuous (episodic) nature of the fictive breathing pattern.
The major results of chapter 5 are as follows: 1) Following the midbrain transection, the effects
of ADO and CCPA on TFV under normocapnic conditions (pH 7.8) were abolished (chapter 5,
figure 5.12). 2) Following midbrain transection, the effects of ADO and CCPA on TFV under
hypercapnic conditions (pH 7.4 and 7.6) remain (chapter 5, figure 5.12). 3) Following the
midbrain transection, the effects of ADO and CCPA on fictive breathing frequency are retained
under hypercapnic conditions (pH 7.4 and 7.6: chapter 5, figure 5.2D) while under normocapnic
conditions (pH 7.8) the effects of CCPA were abolished while the effects of ADO remained but
only during the pH-altering phase of the experiment. 4) Following the midbrain transection the
effect of DPCPX on fictive breathing frequency was abolished due to an increase in the control
level of fictive breathing frequency figures 5.2B and D). 5) Following midbrain transection,
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ADO and CCPA caused increases in fictive breath duration which did not occur prior to the
transection (chapter 5, figure 5.10).
6.2 The Major Conclusions of the Thesis
1) Given that treatment with ADO and CCPA caused decreases in TFV (with the midbrain
intact), the data indicate that activation of A1R causes a decrease in breathing. This is consistent
with the fact that the A1R is an inhibitory receptor. Given that A2 receptors are stimulatory, and
that the A3 receptors are the least expressed sub-type receptor with the lowest affinity for ADO,
it is unlikely that the effects of ADO were mediated by activating receptor sub-types other than
the A1R.
2) Activation of the A1R led to decreases in fictive breathing frequency and may also lead to
increases in fictive integrated breath area although the overall effect is a reduction in TFV. This
is supported by the fact that ADO and CCPA caused a reduction in fictive breathing frequency
while DPCPX caused an increase in fictive breathing frequency but a decrease in fictive
integrated breath area (albeit the effects on area were only observed at pH 7.8 during the
stabilisation period).
3) Activation of the A1 receptor causes a reduction in breathing frequency by reducing the
number of breathing episodes per minute even though A1 receptor stimulation appears to cause a
simultaneous increase in the number of fictive breaths per episode.
4) For the most part, the effects of A1R receptor activation on breathing with the midbrain intact
occur under both normocapnic (pH 7.8) and hypercapnic (pH 7.4 and 7.6) conditions although
the occasional effect is observed at the hypocapnic pH level of 8.0.
5) Adenosine-mediated descending inputs from the rostral region of the midbrain are important
for regulating overall breathing under normocapnic conditions (pH 7.8) but not under
hypercapnic conditions (pH 7.4 and 7.6). This is based on the fact that the effects of ADO and
CCPA on TFV were abolished at pH 7.8 following midbrain transection whereas the effects at
pH 7.4 and 7.6 were not abolished. This does not exclude the possibility or the likelihood that
adenosine-mediated mechanisms in the medullary respiratory centres are also involved in
regulating breathing under normocapnic conditions.
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6) As an extension of the previous conclusion, the data suggest that adenosine-mediated
mechanisms within respiratory centres in the medulla are important for regulating overall
breathing under hypercapnic conditions (pH 7.4 and 7.6) and likely also play a role under
normocapnic conditions. This later point is supported by the fact that the effects of ADO (but not
CCPA) on breathing frequency were retained following the midbrain transection.
7) Adenosine-mediated mechanisms within the medulla normally function to reduce breath
duration but these mechanisms or influences are normally inhibited or reduced by influences
from the midbrain although the nature of these influences is unclear.
Global Conclusion: Activation of A1R receptors causes a reduction in breathing. There are A1R-
mediated mechanisms in the brainstem respiratory centres that reduce breathing under all levels
of respiratory drive (i.e., aCSF pH). In addition, there are A1R-mediated mechanisms within the
midbrain that serve to reduce breathing under normocapnic conditions but not under hypercapnic
conditions.
6.3 The Experimental Approach and Manipulation of Adenosine
Receptors
ADO and A1R analogs (CCPA and DPCPX) were used on intact and midbrain-transected
brainstem preparations to see if the inhibitory modulation on breathing (predominately breathing
frequency) exerted by descending inputs from the midbrain and other rostral brain centers are
mediated by ADO and subsequent activation of the A1R. Administration of ADO and A1R
analogs via circulating aCSF affects all areas of the brainstem preparation. In the intact
brainstem preparations, these include the telencephalon, diencephalon, mesencephalon and
rhombencephalon, whereas in transected brainstem preparations, these include only the
rhombencephalon and the caudal half of the mesencephalon. Hence the difference in breathing in
response to ADO and A1R analogs between intact and midbrain-transected preparations,
described in chapter 5, illustrates how much of the response to ADO and A1R analogs is
accredited to the telencephalon, diencephalon and the rostral half of the mesencephalon.
The data in the current study illustrate that the response to ADO is primarily mediated by the
A1R and that the inhibitory effect of the A1R decreased as the result of the midbrain transection.
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Since the transection removed A1Rs found in the rostral midbrain and rostral brain sites, the
reduction in inhibitory modulation on breathing exerted by the A1R may be due to a reduction in
the overall A1R density in the amphibian brain or simply the specific removal of A1Rs in the
rostral regions of the brain. This finding is consistent with the age-related decrease in the density
of A1R in the brain, which has been demonstrated to decrease the ability of A1Rs to inhibit
neuronal activity (Sebastão et al., 2000) in both mice (Pagonopoulou and Angelatou, 1992) and
humans (Meyer et al., 2007). The current study was also able to demonstrate that antagonizing
the A1R significantly augments fictive breathing frequency in intact brainstem preparations of
the toad, but did not have any effect on fictive breathing frequency and TFV in transected
brainstem preparations. Collectively, the findings suggest that modulation of breathing by ADO
through the A1R is reduced (not eliminated) by the transection, so much so that the activation of
A1R via endogenous ADO does not affect fictive breathing frequency and TFV significantly.
Hence, the hypothesis that the descending inputs from the midbrain and other rostral brain
centers exert an inhibitory modulation on breathing via ADO-mediated mechanisms and the
activation of A1R is supported by these findings.
6.4 The Effects of pH and Adenosine on Respiratory-Related
Motor Output
Neural respiratory-related activity from in vitro brainstem-spinal cord preparations is generated
by respiratory centers found in the medulla and regulated by central pH/CO2 chemoreceptors,
peripheral input from arterial chemoreceptors and lung mechanoreceptors as well as by central
influences from higher brain centers (Lahiri and Forster, 2003; Reid et al., 2000a; Reid, 2006). In
the current study, lowering the aCSF pH (8.0 to 7.4) caused an increase in TFV, fictive breathing
frequency and fictive episodes per minute. This phenomenon, also known as the acute
hypercapnic ventilatory response (or the neural equivalent thereof), is the result of central
pH/CO2 chemoreceptors responding to an increased content of hydrogen (H+) ions caused by the
increase in CO2 that was used to lower the pH of the aCSF (Reid, 2006). In intact animals, CO2
is readily diffusible from the blood and, as such, a rise in CO2 in the cerebrospinal fluid is an
indication of a rise in arterial PCO2 (Kinkead et al., 1994; Lahiri and Forster, 2003; Gheshmy et
al., 2006). Physiologically, hydrogen ions cannot pass through the blood brain barrier, so the
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generation of H+ by the CO2 hydration reaction occurs in the CSF and intracellularly within the
chemoreceptor cells from CO2 as illustrated in the following reaction:
CO2 + H2O →H2CO3 → H+ + HCO3
-
The midbrain-transection in the current study not only maintained the acute hypercapnic
ventilatory response but augmented the sensitivity to elevated CO2 during hypercapnic pH levels
(see figure 5.13A). The maintenance of the acute hypercapnic ventilatory response was expected,
as the medulla and the locus coeruleus (Gargaglioni et al., 2010), which are both sites in the
anuran amphibian brain that contain central pH/CO2 chemoreceptors, were not removed by the
midbrain transection. The increased sensitivity to CO2 is most likely caused by the reduction of
inhibitory modulation through the A1R. In intact and transected brainstem preparations, the
inhibitory modulation of fictive breathing frequency by ADO or CCPA (A1R agonist) was
always observed at hypercapnic pH levels. In addition, treatment with DPCPX (A1R antagonist)
in intact brainstem preparations illustrates that endogenous ADO and subsequent activation of
A1R exert an inhibitory modulation to maintain typical breathing frequency. However,
transecting the brainstem at the level of the midbrain abolished the effect of DPCPX on
breathing frequency, which indicates that the subsequent activation of the A1R by endogenous
ADO no longer had a significant impact on fictive breathing frequency as a result of the
transection.
6.5 Chronic Hypoxia and Adenosine
During a hypoxic event, neurons can become damaged and killed by excitotoxic cell death
(ECD). The primary cause of ECD is the overactivation of NMDA and AMPA receptors which
cause an increase in release of the excitatory neurotransmitter glutamate. When glutamate
increases to excitotoxic levels, substantial amounts of Ca2+
enter into cells which can cause an
increase in cellular activity beyond the cells’ physiological “tolerance” causing the cells to die
(Choi, 1994). However, with an increase in cellular activity there is also an increase in
extracellular ADO due to the heightened use of metabolic by products (Klinger et al., 2002;
Pamenter et al., 2008; Latini and Pedata, 2001). During a hypoxic event, ADO can act as a
retaliatory metabolite by decreasing NMDA and AMPA receptor activity, which inhibits overall
nerve excitability and minimizes neuronal damage caused by ECD (Pamenter et al., 2008).
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Another benefit of ADO during hypoxia is that it helps with the maintenance of brain ATP levels
which are essential to hypoxic tolerance. ADO does this by stimulating ATP production through
anaerobic glycolysis and reducing neuronal energy demands by decreasing neuronal excitability
as well as neurotransmitter release (Bickler and Buck, 2007).
Breathing in the anuran amphibian decreases during chronic hypoxia (McAneney et al., 2006;
McAneney and Reid, 2007). The findings in the current study are generally consistent with the
results observed in toads exposed to chronic hypoxia (CH) in the study by McAneney and Reid
(2007). The attenuation of fictive breathing frequency during CH or in response to perfusion of
ADO results from a decrease in the number of fictive episodes per minute rather than the number
of fictive breaths per episode. The similarity of the data suggests that the reduction in breathing
observed during exposure to CH could be caused by ADO-induced effects on breathing.
However, one notable discrepancy between the two studies does exist; application of 10 μM
ADO or CCPA augmented the number of fictive breaths per episode, an effect that was not
observed following CH. Interestingly, when 1 μM ADO was administered, the augmentation in
the number of fictive breaths per episode was not significant, which may suggest that the number
of breaths clustered into each episode is influenced by the A1R and is concentration dependent.
In future studies, performing microdialysis measurements ADO in the CSF of toads previously
exposed to CH could determine the specific concentration of extracellular ADO achieved in
toads during CH. In addition, administration of DPCPX via cannula to toads in vivo during CH
could determine whether the attenuation to breathing that occurs during CH is mediated by the
A1R.
6.6 Experimental Limitations & Future Suggestions
Westcott scissors were used in the current study to transect the midbrain; a clean cut from the
optic lobes on the dorsal surface to the caudal end of the acuate periventricular nucleus, which
severed the area between the spino-tectal and spino-mesencephalic connections and the dorsal
hypothalamus on the ventral surface (Fig. 2.3). The transection through the midbrain not only
severed modulation from the rostral half of the midbrain, but also from other rostral brain sites,
such as the thalamus, dorsal and ventral hypothalamus, septum, primordial hippocampus,
striatum, etc. In the study by Gargalioni and Branco (2000), electrolytic lesions were made
specifically to the NI located in the midbrain. The results from that study illustrated that the
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lesion removed inhibitory modulation to breathing during elevated respiratory drive but not
under resting conditions, a result which is consistent with the data in the current transection
study. However, the lesions in the study by Gargalioni and Branco (2000) increased breathing
through changes in tidal volume (breath amplitude), but in this thesis the increase in breathing
was achieved through increased breathing frequency and not breath amplitude. The transection in
this thesis is therefore more consistent with the transection done in the study by McAneney and
Reid (2007), as the data from that study also shows an augmentation to breathing during elevated
respiratory drive via an augmentation of fictive breathing frequency rather than fictive breath
amplitude. The difference between the studies could be a reflection of the transection method
used, as both the current study and the study by McAneney and Reid (2007) used Westcott
scissors at a site rostral to the NI to transect the midbrain, whereas Gargaglioni and Branco
(2000) used electrolytic lesions to the NI. Collectively, the findings do not oppose the notion that
the NI may have an inhibitory input to respiratory sites by limiting breathing amplitude when
respiratory drive is elevated. However it still remains uncertain whether the inhibitory input to
respiratory sites that limits fictive breathing frequency during elevated respiratory drive,
demonstrated in the current study and the study by McAneney and Reid (2007), originated from
the midbrain or from other specific brain areas that are rostral to the midbrain. In future studies,
transection of more rostral sites such as the diencephalon or telencephalon would help to isolate
the origin of the inhibitory modulation to elevated respiratory drive.
In intact brainstem preparations, perfusion of ADO onto the brainstem preparation of the toad
had an inhibitory affect on fictive breathing frequency and TFV. The current study was able to
demonstrate that the amount of inhibition to breathing caused by a specific concentration of
ADO can be mirrored with the same concentration of CCPA (A1R agonist), which provides
supporting evidence that the effects of ADO observed in this study were mediated primarily by
the A1R. However, the current study did not test whether the effects of ADO can be mirrored by
stimulation of the A3R, which is also an inhibitory adenosine receptor. In future studies,
perfusion of the A3R agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (CL-
IB-MECA), at the same concentration as that of ADO perfusing the toad brainstem preparation,
could illustrate how much, if any, of the inhibitory effect of ADO on respiration can be
accredited to the A3R.
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Perfusion of DPCPX (A1R antagonist) augmented fictive breathing frequency in intact brainstem
preparations, which illustrates the importance of A1R activation by endogenous ADO in
maintaining typical fictive breathing frequency in the toad. Whether the augmentation to fictive
breathing frequency as a result of DPCPX was caused by antagonizing the A1R alone, or in
combination with endogenous ADO activation of stimulatory A2A and/or A2B receptors, remains
uncertain. In future studies, to determine whether the augmentation of fictive breathing
frequency is mediated by antagonizing the A1R alone, the brainstem preparations could first be
superfused with DPCPX, followed by a wash-out phase and subsequent perfusion of an A2A
antagonist such as SCH-58621 or an A2B antagonist such as imiloxan. Alternatively, perfusion of
DPCPX to the brainstem preparation followed by perfusion of ADO could also determine
whether A1R are fully antagonized and whether the ADO would cause augmentation to breathing
by subsequent activation of stimulatory A2A and/or A2B receptors.
As mentioned previously, the augmentation of fictive breathing frequency seen in intact
preparations in response to DPCPX was not observed in transected brainstem preparations. The
current study demonstrated that A1R modulation was significantly reduced but not eliminated by
the transection. In studies that examined ADO receptor distribution, A2A receptors were found
highly concentrated in the nucleus accumbens, olfactory tubercle, hypothalamus and striatum in
mammals, which were rostral sites that were removed by the transection in the current study.
Hence, there are two possible reasons why the transected preparation no longer responded to
DPCPX: (1) the transection decreased both endogenous ADO sources and A1R modulation to the
point that activation by endogenous ADO no longer impacts fictive breathing, and/or (2) the
transection abolished stimulatory modulation from stimulatory adenosine receptors to fictive
breathing. In future studies, perfusion of A2 agonists (Guanabenz for A2A receptors and BAY
60–6583 for A2B receptor) to transected brainstem preparations should illustrate whether
stimulatory modulation through A2 receptors is still intact or removed by the transection.
Alternatively, utilizing RT-PCR experiments or an in vitro light microscopic autoradiographic
method (Goodman and Snyder, 1982) could help verify the relative densities of ADO receptors
in the brain of cane toad.
100
6.7 Conclusion
Activation of A1R receptors causes a reduction in breathing. There are A1R-mediated
mechanisms in the brainstem respiratory centres that reduce breathing under all levels of
respiratory drive (i.e., aCSF pH). In addition, there are A1R-mediated mechanisms within the
midbrain that serve to reduce breathing under normocapnic conditions but not under hypercapnic
conditions.
101
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Appendix
113
A1. Summary of the Experimental Protocol and an Explanation of the Data Contained Within the Appendix
Figure 3.1: Overview of the stages and general procedures performed within the experiments on intact brainstem-
spinal cord preparations.
Figure 5.1: Overview of the stages and general procedures used within the experiment on midbrain transected
preparations.
The two diagrams above (Figures 3.1 and 5.1) are reproduced from chapters 3 and 5,
respectively. Details can be found in the appropriate figure legends within these chapters. The
data reported within chapters 3, 4 and 5 of the thesis were all recorded during the “post-
treatment” phase of the experiments. All pre-treatment values (i.e. prior to experimental
manipulation – pharmaceutical agent administration and/or midbrain transection) are reported
within this appendix. The pre-treatment phase of the experiment was split into two parts; (1) the
stabilisation period: circulating virgin aCSF at constant pH of 7.8 and (2) the pH-treatment
(varying) period: alterations made to aCSF pH to achieve values of 7.4, 7.6, 7.8 and 8.0. During
114
the pre-treatment phase of the experiment, the in vitro brainstem-spinal cord preparations were
intact and were treated with circulating virgin aCSF only.
Data pertaining to the effects of time (i.e., during the time course of the entire experimental
protocol) are included within the appendix. To evaluate whether time had any effect on the
results, values from all the respiratory variables are reported from all four stages of the
experiment (Fig. 2.2; reproduced below) at a pH of 7.8 only in the intact control groups. These
data will show (see below) that all measured variables at pH 7.8 in control preparations with the
midbrain intact are identical throughout the four experimental stages of the protocol. This
indicates that “time alone” had no confounding effect on the results. Indeed this is consistent
with the long-held knowledge that the isolated brainstem-spinal cord preparation from
amphibians is robust and can function normally for many hours (and in some cases several days).
In addition, a series of recordings from midbrain transected preparations are included in this
appendix to illustrate that continuous fictive breathing did occur as a result of the transection,
albeit temporarily, reverting back to discontinuous (episodic) fictive breathing usually before the
end of the transection stabilisation period.
115
Figure 2.2. Flow chart illustrating the general stages of the experimental protocol used in the various series of
experiments outlined in this thesis. The first step was the stabilisation period, where the brainstem preparation was
placed into a bath of circulating aCSF at constant pH 7.8 until fictive breathing became rhythmic for at least 15
minutes. The second step was the pH treatment period, where the brainstem preparation was subjected to random
alterations of aCSF pH (7.4, 7.6, 7.8 and 8.0). The third step was the experimental manipulation period in which a
dose of adenosine, CCPA or DPCPX was introduced into the aCSF superfusing the brainstem preparations that were
either kept intact or transected at the midbrain. The fourth step was the “dose and pH treatment period”, where the
brainstem preparation was exposed to random alterations in aCSF in the continued presence of adenosine, CCPA or
DPCPX.
116
A2. Data Analysis
A one-way repeated measures analysis of variance followed by a Holm-Sidak multiple
comparison test was used to analyze the effect of altering aCSF pH levels within each treatment
group; the value obtained for each respiratory variable at pH 7.4, 7.6 and 8.0 was compared to
the value of the respective respiratory variable obtained at the control pH of 7.8. A one-way
repeated measures analysis of variance was also used to evaluate fictive breathing during the
stabilisation period by comparing the value of the respective variable between the control groups
to a specific group designated for a specific treatment. To evaluate the effects of time, a one-way
repeated analysis of variance was used to compare respective respiratory variables obtained at
each stage of the experiment to one other. To evaluate the effect of both the dose and pH, a two-
way ANOVA with the Holm-Sidak method was used to compare the values of the respiratory
variable obtained at pH 7.4, 7.6, 7.8 and 8.0 in control groups to the values of the respective
respiratory variable and relative pH set in a group designated for a specific treatment. The limit
of significance was 5% (p < 0.05), and all data was expressed as the mean ± one standard error
of the mean (S.E.M).
117
A3. Results
A3.1 Fictive Breathing Frequency
Figure A.1: Fictive breathing frequency (fictive breaths per minute) measured during the pre-treatment phase of the
experiment in all groups examined within the current study. (A) The fictive breathing frequency values observed in
all groups was the same (no statistically-significant differences) during the stabilisation period of the pre-treatment
phase. (B) Fictive breathing frequency within all groups progressively decreased as aCSF pH was increased during
the pH treatment (pH-varying) period of the pre-treatment phase and the values in all groups were the same (no
statistically-significant differences). The grey area in panel A represents treatment groups that were subjected to a
midbrain transection later on in the study. The data are reported as the mean ± S.E.M.
118
A3.2 Fictive Episodes per Minute
Figure A.2: The number of fictive breathing episodes per minute measured during the pre-treatment phase of the
experiment in all groups examined within the current study. (A) The fictive breathing frequency values observed in
all groups was the same (no statistically-significant differences) during the stabilisation period of the pre-treatment
phase. (B) Fictive breathing frequency within all groups progressively decreased as aCSF pH was increased during
the pH treatment (pH-varying) period of the pre-treatment phase and the values in all groups were the same (no
statistically-significant differences). The grey area in panel A represents treatment groups that were subjected to a
midbrain transection later on in the study. The data are reported as the mean ± S.E.M.
119
A3.3 Fictive Breaths per Episode
Figure A.3: The number of fictive breaths per episode measured during the pre-treatment phase of the experiment in
all groups examined within the current study. (A) The fictive breathing frequency values observed in all groups was
the same (no statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B)
Fictive breathing frequency within all groups progressively decreased as aCSF pH was increased during the pH
treatment (pH-varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-
significant differences). The grey area in panel A represents treatment groups that were subjected to a midbrain
transection later on in the study. The data are reported as the mean ± S.E.M.
120
A3.4 Total Fictive Ventilation Index
Figure A.4: Total fictive ventilation measured during the pre-treatment phase of the experiment in all groups
examined within the current study. (A) The fictive breathing frequency values observed in all groups was the same
(no statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B) Fictive
breathing frequency within all groups progressively decreased as aCSF pH was increased during the pH treatment
(pH-varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-
significant differences). The grey area in panel A represents treatment groups that were subjected to a midbrain
transection later on in the study. The data are reported as the mean ± S.E.M.
121
A3.5 Integrated Area of the Fictive Breaths
Figure A.5: Fictive breath area measured during the pre-treatment phase of the experiment in all groups examined
within the current study. (A) The fictive breathing frequency values observed in all groups was the same (no
statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B) Fictive breathing
frequency within all groups progressively decreased as aCSF pH was increased during the pH treatment (pH-
varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-significant
differences). The grey area in panel A represents treatment groups that were subjected to a midbrain transection later
on in the study. The data are reported as the mean ± S.E.M.
122
A3.6 Fictive Breath Duration
Figure A.6: Fictive breath duration measured during the pre-treatment phase of the experiment in all groups
examined within the current study. (A) The fictive breathing frequency values observed in all groups was the same
(no statistically-significant differences) during the stabilisation period of the pre-treatment phase. (B) Fictive
breathing frequency within all groups progressively decreased as aCSF pH was increased during the pH treatment
(pH-varying) period of the pre-treatment phase and the values in all groups were the same (no statistically-
significant differences). The grey area in panel A represents treatment groups that were subjected to a midbrain
transection later on in the study. The data are reported as the mean ± S.E.M.
123
A3.7 Time
Figure A.7: The effect of time on respiratory variables recorded from intact control brainstem-spinal cord
preparations. (A) Fictive breathing frequency, (B) total fictive ventilation, (C) the number of fictive episodes per
minute, (D) fictive breath area, (E) the number of fictive breaths per episode and (F) fictive breath duration. Stage 1
represents the pre-treatment stabilisation phase; Stage 2 represents the pre-treatment pH treatment (pH-varying)
phase; Stage 3 represents the post-treatment stabilisation phase; Stage 4 represents the post-treatment dose & pH
treatment phase (pH-varying phase). All of the data were recorded at pH 7.8 at each stage. With the exception of
fictive breath duration in stage 3(#), the values for each variable recorded at pH 7.8 during the four experimental
stages were not statistically different. The data are reported as the mean ± S.E.M.
124
A3.8 Midbrain Transection
Figure A.8: Fictive breathing (trigeminal motor output) recorded at pH 7.8 before and after a midbrain transection.
(A) A recording made during the pre-treatment stabilisation phase (“pre-midbrain transection”). (B) A recording
during the post-treatment (i.e., post-transection) stabilisation period (“transection”). (C) A recording made during
the post-treatment (i.e., post-transection) pH-treatment (pH-varying) phase. Large peaks represent fictive lung
breaths and small peaks represent fictive buccal oscillations. In all cases the upper trace represents the raw
electroneurogram (eng X) recorded from the trigeminal nerve root while the lower trace (∫eng X) represents the
integrated trace.
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